Antisense oligonucleotides for treatment of cancer stem cells

ABSTRACT

The invention provides oligonucleotides complementary to a non-coding chimeric mitochondrial RNA as well as compositions and kits comprising the same, and their use in treating and preventing metastasis or relapse of a cancer in an individual previously treated for cancer with a therapy. The invention also provides oligonucleotides complementary to a non-coding chimeric mitochondrial RNA as well as compositions and kits comprising the same, and their use in treating a refractory cancer (e.g., a refractory HPV-associated cancer).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 61/785,269, filed Mar. 14, 2013, U.S. ProvisionalPatent Application Ser. No. 61/790,072, filed Mar. 15, 2013, and U.S.Provisional Patent Application Ser. No. 61/937,438, filed Feb. 7, 2014,the entire content of each of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to oligonucleotides complementary to a non-codingchimeric mitochondrial RNA and their use in methods of treating andpreventing metastasis or relapse of a cancer in an individual previouslytreated for cancer. The invention also relates to oligonucleotidescomplementary to a non-coding chimeric mitochondrial RNA and their usein treating a refractory cancer (e.g., a refractory HPV associatedcancer) in an individual.

BACKGROUND OF THE INVENTION

Cancer is a cellular malignancy whose unique trait, loss of normalcontrol of cell cycle, results in unregulated growth, lack ofdifferentiation, and ability to invade other tissues and metastasize.Carcinogenesis is a multi-step process by which a normal cell istransformed in a malignant cell (McKlima et al., “The Biology Basis ofCancer”, Ch. 3, 1998). The etiology of cancer is complex and includesalteration of the cell cycle regulation, chromosomal abnormalities andchromosomes breakage. Infectious agents (e oncogenic viruses),chemicals, radiation (e.g., ultraviolet or ionizing radiation) andimmunological disorders are thought to be the major causes ofcarcinogenesis (McKinnell et al., “The Biological Basis of Cancer, Ch.3, 1998).

Recent evidence supports the view that tumors are organized in ahierarchy of heterogeneous cell populations with different biologicalproperties. Two models have been proposed to account for thisheterogeneity within tumors and for tumor growth. One such model isbased on cancer stem cells (CSCs) which are thought to be responsiblefor aspects of cancer such as initiation, progression, metastasis, andrecurrence. See Chen et al., Acta Pharmacol Sin., 34(6):732-740, 2013;Ponti et al., Cancer Res, 65(13):5506-11, 2005; Singh et al., CancerRes. 63:5821-5828, 2003; and Feng et al., Oncology Reports,22:1129-1134, 2009, Although CSCs generally represent a very smallpopulation of the overall tumor population, they are generally regardedas a self-renewing initiation subpopulation of tumor cells or a smallpopulation of cancer cells that are capable of giving rise to newtumors. CSCs have been identified in a number of cancers including, butnot limited to, breast, brain, blood, liver, kidney, cervical, ovarian,colon, and lung cancers among others. See Ponti et al., Cancer Res,65(13):5506-11, 2005; Feng et al., Oncology Reports, 22:1129-1134, 2009;Zhang et al., Cancer Res, 68(10:4311:4320, 2008; Singh et al., CancerRes, 63:5821-5828, 2003; Clarke et al., Cancer Res, 66:9339, 2006;Sendurai et al., Cell, 133:704, 2008; Ohata et al., Cancer Res, 72:5101,2012; and Mukhopapadhyay et al., Plos One, 8(11):e78725, 2013).

Surgical resection of tumor(s) or metastases arising from a primarytumor(s) followed by systemic administration of anti-cancer therapy isthe established clinical protocol for treatment of several cancers.Although successful for treatment of some cancer types, a well-knowncomplication of cancer treatment is the survival of residual tumor cellsor CSCs that are not effectively removed which can result in relapseafter remission, with the cancer returning at the primary site of tumorformation or at distant sites due to metastasis. Recently, it was foundthat CSCs may also contribute to relapse after remission due toresistance to chemotherapy. See Domingo-Domenech et al., Cancer Cell,22(3):373, 2012. Therefore, there is a need for development oftherapeutic agents that can target cells which contribute to relapse andmetastasis (e.g., CSCs). Discovery of such therapeutic agents may allowfor the development of treatment useful for preventing, recurrence ofcancer after remission (i.e., relapse) or preventing the spread of theprimary tumour to secondary sites (i.e., metastasis). See Clarke et al.,Cancer Res, 66:9339, 2006.

All references cited herein, including patent applications, patentpublications, and scientific literature, are herein incorporated byreference as if each individual reference were specifically andindividually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

The invention provided herein discloses, inter alia, methods forsuppressing metastasis of a cancer in an individual comprisingadministering to the individual an effective amount of one or moreoligonucleotide complementary to an antisense non-coding chimericmitochondrial RNA (ASncmtRNA) molecule or a sense non-coding chimericmitochondrial RNA (SncmtRNA) molecule, wherein the oligonucleotide isable to hybridize with the chimeric mitochondrial RNA molecules to forma stable duplex, and wherein the individual has been previously treatedfor cancer with a therapy. In some embodiments, the oligonucleotide issufficiently complementary to a human non-coding chimeric mitochondrialRNA molecule comprising: an antisense 16S mitochondrial ribosomal RNAcovalently linked at its 5′ end to the 3′ end of a polynucleotide withan inverted repeat sequence or a sense 16S mitochondrial ribosomal RNAcovalently linked at its 5′ end to the 3′ end of a polynucleotide withan inverted repeat sequence. In any of the embodiments herein, theoligonucleotide can be complementary to the ASncmtRNA molecule encodedby a nucleotide sequence selected from the group consisting of SEQ IDNO:4, SEQ NO:5, and SEQ ID NO:6. In any of the embodiments herein, theoligonucleotide can be at least 85% complementary to the ASncmtRNAmolecule encoded by a nucleotide sequence selected from the groupconsisting SEQ NO:4, SEQ ID NO:5, and SEQ ID NO:6. In any of theembodiments herein, the one or more oligonucleotide can comprise anucleotide sequence selected from the group consisting of SEQ NOs:7-198.In some embodiments, the one or more, oligonucleotide comprises anucleic acid sequence selected from the group consisting of SEQ IDNOs:36, 197 and 198. In any of the embodiments herein, theoligonucleotide can be administered in combination with at least oneanti-cancer agent. In a further embodiment, the at least one anti-canceragent is selected from the group consisting of remicade, docetaxel,celecoxib, melphalan, dexamethasone, steroids, gemcitabine, cisplatinum,temozolomide, etoposide, cyclophosphamide, temodar, carboplatin,procarbazine, gliadel, tamoxifen, topotecan, methotrexate, gefitinib,taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11,interferon alpha, pegylated interferon alpha, capecitabine, cisplatin,thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine,doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine,vinorelbine, zoledronic acid, palmitronate, biaxin, busulphan,prednisone, bortezomib, bisphosphonate, arsenic trioxide, vincristine,doxoruhicin, paclitaxel, ganciclovir, adriamycin, estrainustine sodiumphosphate, sulindac, and etoposide. In some embodiments, theoligonucleotide and the at least one anti-cancer agent is administeredsequentially. In some embodiments, the oligonucleotide and the at leastone anti-cancer agent is administered simultaneously. In any of theembodiments herein, the oligonucleotide can be administered incombination with a radiation therapy. In any of the embodiments herein,the oligonucleotide can be administered in combination with surgery. Inany of the embodiments herein, the oligonucleotide can be administeredin combination with an allogenic stem cell transplant therapy. In any ofthe embodiments herein, the oligonucleotide can be administered incombination with an autologous stem cell transplant therapy. In any ofthe embodiments herein, the individual may have been previously treatedfor cancer with a therapy comprising chemotherapy, radiation therapy,surgery, or combinations thereof. In any of the embodiments herein, thecancer in the individual may have relapsed after treatment with one ormore of bortezomib, cyclophosphamide, dexamethasone, doxorubicin,interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha,prednisone, thalidomide, and vincristine. In any of the embodimentsherein, wherein the cancer can be a solid cancer. In a furtherembodiment, the solid cancer is bladder cancer, brain cancer, breastcancer, cervical cancer, colon cancer, endometrial cancer, esophagealcancer, gastric cancer, liver and bile duct cancer, lung cancer,melanoma, oral cancer, ovarian cancer, pancreatic cancer, pharynxcancer, prostate cancer, renal cancer, testicular cancer, or thyroidcancer. In any of the embodiments herein, wherein the cancer can be anon-solid cancer. In a further embodiment, the non-solid cancer ismultiple myeloma, leukemia, or lymphoma. In any of the embodimentsherein, the oligonucleotide can reduce the number of cancer stem cellsin the individual as compared to an individual not administered theoligonucleotide. In any of the embodiments herein, the oligonucleotidecan inhibit tumor growth and/or metastasis in the individual as comparedto an individual not administered the oligonucleotide.

In one aspect, the invention provided herein discloses, methods forpreventing relapse of cancer in an individual comprising administeringto the individual an effective amount of one or more oligonucleotidecomplementary to an antisense non-coding chimeric mitochondrial RNA(ASncmtRNA) molecule or a sense non-coding chimeric mitochondrial RNA(SncmtRNA) molecule, wherein the oligonucleotide is able to hybridizewith the chimeric mitochondrial RNA molecules to form a stable duplex,and wherein the individual has been previously treated for cancer with atherapy. In a further embodiment, the oligonucleotide is sufficientlycomplementary to a human non-coding chimeric mitochondrial RNA moleculecomprising: an antisense 16S mitochondrial ribosomal RNA covalentlylinked at its 5′ end to the 3′ end of a polynucleotide with an invertedrepeat sequence or a sense 16S mitochondrial ribosomal RNA covalentlylinked at its 5′ end to the 3′ end of a polynucleotide with an invertedrepeat sequence. In any of the embodiments herein, the oligonucleotidecan be complementary to the ASncmtRNA molecule encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5,and SEQ ID NO:6. In any of the embodiments herein, the oligonucleotidecan be at least 85% complementary to the ASncmtRNA molecule encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:4,SEQ ID NO:5, and SEQ ID NO:6. In any of the embodiments herein, the oneor more oligonucleotide can comprise a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:7-198. In some embodiments, the oneor more oligonucleotide comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:36, 197 and 198. In any of theembodiments herein, the oligonucleotide can be administered incombination with at least one anti-cancer agent. In a furtherembodiment, the at least one anti-cancer agent is selected from thegroup consisting of remicade, docetaxel, celecoxib, melphalan,dexamethasone, steroids, gemcitabine, cisplatinum., temozolomide,etoposide, cyclophosphamide, temodar, carboplatin, procarbazine,gliadel, tamoxifen, topotecan, methotrexate, gefitinib, taxol, taxotere,fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha,pegylated interferon alpha, capecitabine, cisplatin, thiotepa,fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol,pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine,zoledronic acid, palmitronate, biaxin, busulphan, prednisone,bortezomib, bisphosphonate, arsenic trioxide, vincristine, doxorubicin,paclitaxel, ganciclovir, adriamycin, estrainustine sodium phosphate,sulindac, and etoposide. In some embodiments, the oligonucleotide andthe at least one anti-cancer agent is administered sequentially. In someembodiments, the oligonucleotide and the at least one anti-cancer agentis administered simultaneously. In any of the embodiments herein, theoligonucleotide can be administered in combination with a radiationtherapy. In any of the embodiments herein, the oligonucleotide can headministered in combination with surgery. In any of the embodimentsherein, the oligonucleotide can be administered in combination with anallogenic stem cell transplant therapy. In any of the embodimentsherein, the oligonucleotide can be administered in combination with anautologous stem cell transplant therapy. In any of the embodimentsherein, the individual may have been previously treated for cancer witha therapy comprising chemotherapy, radiation therapy, surgery, orcombinations thereof. In any of the embodiments herein, the cancer inthe individual may have relapsed after treatment with one or more ofbortezomib, cyclophosphamide, dexamethasone, doxorubicin,interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha,prednisone, thalidomide, and vincristine. In any of the embodimentsherein, wherein the cancer can be a solid cancer. In a furtherembodiment, the solid cancer is bladder cancer, brain cancer, breastcancer, cervical cancer, colon cancer, endometrial cancer, esophagealcancer, gastric cancer, liver and bile duct cancer, lung cancer,melanoma, oral cancer, ovarian cancer, pancreatic cancer, pharynxcancer, prostate cancer, renal cancer, testicular cancer, or thyroidcancer. In any of the embodiments herein, wherein the cancer can be anon-solid cancer. In a further embodiment, the non-solid cancer ismultiple myeloma, leukemia, or lymphoma. In any of the embodimentsherein, the oligonucleotide can reduce the number of cancer stem cellsin the individual as compared to an individual not administered theoligonucleotide. In any of the embodiments herein, the oligonucleotidecan inhibit tumor growth and/or metastasis in the individual as comparedto an individual not administered the oligonucleotide.

In yet another aspect, the invention provided herein discloses, methodsfor the treatment of metastatic cancer in an individual comprisingadministering to the individual an effective amount of one or moreoligonucleotide complementary to an antisense non-coding chimericmitochondrial RNA (ASncmtRNA) molecule or a sense non-coding chimericmitochondrial RNA (SncmtRNA) molecule, wherein the oligonucleotide isable to hybridize with the chimeric mitochondrial RNA molecules to forma stable duplex, and wherein the individual has been previously treatedfor cancer with a therapy. In a further embodiment, the oligonucleotideis sufficiently complementary to a human non-coding chimericmitochondrial RNA molecule comprising: an antisense 16Smitochondria(ribosomal RNA covalently linked at its 5′ end to the 3′ endof a polynucleotide with an inverted repeat sequence or a sense 16Smitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′end of a polynucleotide with an inverted repeat sequence. In any of theembodiments herein, the oligonucleotide can be complementary to theASncmtRNA molecule encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In any ofthe embodiments herein, the oligonucleotide can be at least 85%complementary to the ASncmtRNA molecule. encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5,and SEQ ID NO:6. In any of the embodiments herein, the one or moreoligonucleotide can comprise a nucleotide sequence selected from thegroup consisting of SEQ ID NOs:7-198. In some embodiments, the one ormore oligonucleotide comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:36, 197 and 198. In any of theembodiments herein, the oligonucleotide can be administered incombination with at least one anti-cancer agent. In a furtherembodiment, the at least one anti-cancer agent is selected from thegroup consisting of remicade, docetaxel, celecoxib, melphalan,dexamethasone, steroids, gemcitabine, cisplatinum, temozolomide,etoposide, cyclophosphamide, temodar, carboplatin, procarbazine,gliadel, tamoxifen, topotecan, methotrexate, gefitinib, taxol, taxotere,fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha,pegylated interferon alpha, capecitabine, cisplatin, thiotepa,fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol,pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine,zoledronic acid, palmitronate, biaxin, busulphan, prednisone,bortezomib, bisphosphonate, arsenic trioxide, vincristine, doxorubicin,paclitaxel, ganciclovir, adriamycin, estrainustine sodium phosphate,sulindac, and etoposide. In some embodiments, the oligonucleotide andthe at least one anti-cancer agent is administered sequentially. In someembodiments, the oligonucleotide and the at least one anti-cancer agentis administered simultaneously. In any of the embodiments herein, theoligonucleotide can be administered in combination with a radiationtherapy. In any of the embodiments herein, the oligonucleotide can beadministered in combination with surgery. In any of the embodimentsherein, the oligonucleotide can be administered in combination with anallogenic stem cell transplant therapy. In any of the embodimentsherein, the oligonucleotide can be administered in combination with anautologous stem cell transplant therapy. In any of the embodimentsherein, the individual may have been previously treated for cancer witha therapy comprising chemotherapy, radiation therapy, surgery, orcombinations thereof. In any of the embodiments herein, the metastaticcancer in the individual may have relapsed after treatment with one ormore of bortezomib, cyclophosphamide, dexamethasone, doxorubicin,interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha,prednisone, thalidomide, and vincristine. In any of the embodimentsherein, wherein the cancer can be a solid cancer. In a furtherembodiment, the solid cancer is bladder cancer, brain cancer, breastcancer, cervical cancer, colon cancer, endometrial cancer, esophagealcancer, gastric cancer, liver and bile duct cancer, lung cancer,melanoma, oral cancer, ovarian cancer, pancreatic cancer, pharynxcancer, prostate cancer, renal cancer, testicular cancer, or thyroidcancer. In any of the embodiments herein, wherein the cancer can be anon-solid cancer. In a further embodiment, the non-solid cancer ismultiple myeloma, leukemia, or lymphoma. In any of the embodimentsherein, the oligonucleotide can reduce the number of cancer stem cellsin the individual as compared to an individual not administered theoligonucleotide. In any of the embodiments herein, the oligonucleotidecan inhibit tumor growth and/or metastasis in the individual as comparedto an individual not administered the oligonucleotide.

In yet another aspect, the invention provided herein discloses, methodsfor the treatment of a refractory cancer (e.g., a refractoryHPV-associated cancer) in an individual comprising administering to theindividual an effective amount of one or more oligonucleotidecomplementary to an antisense non-coding chimeric mitochondrial RNA(ASncmtRNA) molecule or a sense non-coding chimeric mitochondria' RNA(SncmtRNA) molecule, wherein the oligonucleotide is able to hybridizewith the chimeric mitochondrial RNA molecules to form a stable duplex.In a further embodiment, the oligonucleotide is sufficientlycomplementary to a human non-coding chimeric mitochondrial RNA moleculecomprising: an antisense 16S mitochondrial ribosomal RNA covalentlylinked at its 5′ end to the 3′ end of a polynucleotide with an invertedrepeat sequence or a sense 16S mitochondrial ribosomal RNA covalentlylinked at its 5′ end to the 3′ end of a polynucleotide with an invertedrepeat sequence. In any of the embodiments herein, the oligonucleotidecan be complementary to the ASncmtRNA molecule encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5,and SEQ ID NO:6. In any of the embodiments herein, the oligonucleotidecan be at least 85% complementary to the ASncmtRNA molecule encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:4,SEQ ID NO:5, and SEQ ID NO:6. In any of the embodiments herein, the oneor more oligonucleotide can comprise a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:7-198. In some embodiments, the oneor more oligonucleotide comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:36, 197 and 198. In any of theembodiments herein, the oligonucleotide can be administered incombination with at least one anti-cancer agent. In some embodiments,the oligonucleotide and the at least one anti-cancer agent isadministered sequentially. In some embodiments, the oligonucleotide andthe at least one anti-cancer agent is administered simultaneously. Inany of the embodiments herein, the oligonucleotide can be administeredin combination with a radiation therapy. In any of the embodimentsherein, the oligonucleotide can be administered in combination withsurgery. In any of the embodiments herein, the oligonucleotide canreduce the number of cancer stem cells in the individual as compared toan individual not administered the oligonucleotide. In any of theembodiments herein, the oligonucleotide can inhibit tumor growth and/ormetastasis in the individual as compared to an individual notadministered the oligonucleotide.

In another aspect, the invention herein provides kits comprising one ormore oligonucleotide complementary to an antisense non-coding chimericmitochondrial RNA (ASncmtRNA) molecule or a sense non-coding chimericmitochondrial RNA (SncmtRNA) molecule and instructions for practicingany method disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a general scheme of the experimental procedure and theassay utilized herein to measure the effect of antisenseoligonucleotides targeted to ASncmtRNA un the number of spheres formedin colon cancer cells, based on the specific ability of cancer stemcells to form these spheroid bodies.

FIG. 2 depicts representative examples of spheres formed in primarycolon tumor cancer cells following no treatment, treatment with onlyLipofectamine, treatment with a control oligonucleotide (Control Oligo154), or treatment with an antisense oligonucleotide (ASO) targeted toASncmtRNA (ASO 1537S). Treatment with the ASO 1537S abolished theformation of spheres.

FIG. 3 depicts a quantification of the capacity of primary colon tumorcells to form spheres. Approximately 0.6% of the total number of cellsseeded were able to form spheres. Cells transfected with ASO 1537S wereunable to form spheres.

FIG. 4 depicts representative examples of spheres formed in cells fromthe HCT-116 colon cancer cell line following no treatment, treatmentwith only Lipofectamine, treatment with a control oligonucleotide(Control Oligo 154), or treatment with an antisense oligonucleotidetargeted to ASncmtRNA (ASO 1107S). Treatment with ASO 1107S abolishedthe formation of spheres.

FIG. 5 depicts a quantification of the capacity of HCT-116 colon cancercells to form spheres. Approximately 0.3% of the total number of cellsseeded were able to form spheres. Cells transfected with ASO 1107S wereunable to form spheres.

FIG. 6 depicts a general scheme of the experimental procedure and theassay utilized herein to measure the effect of antisenseoligonucleotides targeted to ASncmtRNA on the number of spheres formedby the cervical cancer SiHa cell line and primary culture cells ofcervical tumors. The assay is based on the ability of cancer stem cellsto form spheres, also referred to herein as spheroid bodies.

FIG. 7 depicts representative examples of spheres formed by the cervicalcancer SiHa cell line and primary culture cells of cervical tumorsfollowing no treatment (NT), treatment with a control oligonucleotide(Control Oligo 154: ASO-C), or treatment with an antisenseoligonucleotide targeted to ASncmtRNA (ASO 1537S) or treatment with 45μM cisplatin (CISP). Treatment with the ASO 1537S abolished theformation of spheres. The CerCa 3 cells obtained from primary culture,which is infected with HPV 45, is resistant to treatment with cisplatinas compared to two other cells obtained from primary culture, which areinfected with HPV 16.

FIG. 8 depicts quantification of sphere formation by the cell cell lineand primary cervical tumor cells (CerCa 1, CerCa 2 and CerCa 3) with orwithout treatment. Cells transfected with ASO 1537S wer unable to formspheres. The CerCa 3 primary culture infected with HPV 45 was resistantto treatment with cisplatin but not to treatment with ASO 1537S.

FIG. 9 depicts the absence of tumor relapse and the absence ofmetastatic nodules in the lungs and liver of mice treated with ASO 1560S(squares) but not Control Oligo 154 (circles) following the surgicalremoval of intradermal melanoma tumors.

FIG. 10 depicts the presence of tumor relapse metastatic black nodulesin the lung and livers of the mice treated with Control Oligo 154 butnot in the lungs and livers mice treated with ASO 1560S.

FIG. 11 depicts the absence of tumor relapse and complete survival ofmice treated with ASO 1560S (triangles) but not Control ASO 154(squares) following surgical removal of intradermal kidney carcinomatumors.

FIG. 12 depicts the absence of relapse in and survival of mice treatedwith ASO 1560S (squares) but not Control ASO 154 (circles) followingsurgical removal of intradermal kidney carcinoma tumors.

FIG. 13 depicts the absence of tumor relapse and complete survival ofmice treated with ASO 1560S but not Control ASO 154 following surgicalremoval of intradermal melanoma carcinoma tumors.

FIG. 14 depicts the absence of tumors and complete survival of micetreated with ASO 1560S but not Control ASO 154 following surgicalremoval of subcutaneous bladder carcinoma tumors. ip indicatesintraperitoneal administration and iv indicates intravenousadministration.

FIG. 15 depicts the reduction in tumors and increase in survival of Rag-/- mice treated with ASO 11537S but not Control ASO 154 following theremoval of a human A375 melanoma tumor.

DETAILED DESCRIPTION

The invention provided herein discloses, infer cilia, compositionscomprising one or more oligonucleotide complementary to an antisensenon-coding chimeric mitochondrial RNA (ASncmtRNA) molecule or a sensenon-coding chimeric mitochondrial RNA (SncmtRNA) molecule and usesthereof for suppressing metastasis of a cancer in an individual. Incertain embodiments, the invention provides compositions comprising oneor more oligonucleotide complementary to an ASncmtRNA molecule or aSncmtRNA molecule and uses thereof for treating or preventing relapse ofa cancer in an individual. In certain embodiments, the inventionprovides compositions comprising one or more oligonucleotidecomplementary to an ASncmtRNA molecule or a SncmtRNA molecule and usesthereof for treating metastatic cancer in an individual. In certainembodiments, the invention provides compositions comprising one or moreoligonucleotide complementary to an ASncmtRNA molecule or a SncmtRNAmolecule and uses thereof for treating a refractory cancer (e.g., arefractory HPV-associated cancer) in an individual. In some of theembodiments herein, the individual has been previously treated forcancer with a therapy (e.g., chemotherapy, radiation therapy, surgery orcombinations thereof).

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, biochemistry, nucleic acid chemistry, and immunology,which are well known to those skilled in the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); PCR: The Polymerase Chain Reaction, (Mulliset al., eds.. 1994); Harlow and Lane (1988) Antibodies. A LaboratoryManual, Cold Spring Harbor Publications, New York; Harlow and Lane(1999) Using Antibodies: A Laboratory Manual Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (jointly referred to hereinas “Harlow and Lane”), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000),Handbook of Experimental Immunology, 4th edition (D, M. Weir & C. C.Blackwell, eds., Blackwell Science Inc., 1987); and Gene TransferVectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987).Other useful references include Harrison's Principles of InternalMedicine (McGraw Hill; J. Isseleacher et al., eds.), Dubois' LupusErythematosus (5th ed.; D. J. Wallace and B. H. Hahn eds.

II. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. Thus, for example, reference to“an oligonucleotide” optionally includes a combination of two or moresuch oligonucleotides, and the like.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

An “isolated” nucleic acid molecule (e.g., “isolated oligonucleotide”)is a nucleic acid molecule that is identified and separated from atleast one contaminant nucleic acid molecule with which it is ordinarilyassociated in the natural source of the nucleic acid. An isolatednucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated nucleic acid molecules therefore aredistinguished from the nucleic acid molecule as it exists in naturalcells,

As used herein, the term “oligonucleotide complementary to an antisensenon-coding chimeric mitochondrial RNA” or “oligonucleotide complementaryto a sense non-coding chimeric mitochondrial RNA” refers to a nucleicacid having sufficient sequence complementarity to a target antisensenon-coding chimeric mitochondrial RNA or a target sense non-codingchimeric mitochondrial RNA, respectively. An oligonucleotide“sufficiently complementary” to a target antisense non-coding chimericmitochondrial RNA or a target sense non-coding chimeric mitochondrialRNA means that the oligonucleotide has a sequence sufficient tohybridize with the chimeric mitochondrial RNA molecules to form a stableduplex.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. An “oligonucleotide composition” of theinvention includes any agent, compound or composition that contains oneor more oligonucleotides, and includes, e.g., compositions comprisingboth single stranded and/or double stranded (ds) oligonucleotides,including, e.g., single stranded RNA, single stranded DNA, DNA/DNA andRNA/DNA hybrid oligonucleotides, as well as derivatized/modifiedoligonucleotides thereof. Such “oligonucleotide compositions” may alsoinclude amplified oligonucleotide products, e.g., polymerase chainreaction (PCR) products. An “oligonucleotide compositions” of theinvention may also include art-recognized compositions designed to mimicthe activity of oligonucleotides, such as peptide nucleic acid (PNA)molecules.

The phrase “corresponds to” or “sequence corresponding to” as it relatesto RNA described herein (e.g., ASncmtRNA), indicates that the RNA has asequence that is identical to or substantially the same as an RNA, or anRNA encoded by an analogous DNA, described herein. For example, anASncmtRNA that corresponds to SEQ ID NO:4 indicates that the ASncmtRNAhas a sequence that is identical to or substantially the same as the RNAof SEQ ID NO:203 or the RNA encoded by the analogous DNA of SEQ ID NO:4.

“Percent (%) nucleic acid sequence identity” or “percent (%)complementary” with respect to a reference nucleotide sequence (e.g.,SncmtRNA sequence or ASncmtRNA sequence) is defined as the percentage.of nucleic acid residues in a candidate sequence (e.g., oligonucleotidesequence) that are identical with the nucleic acid residues in thereference nucleotide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentnucleic acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For example, the % nucleic acid sequence identity of a given nucleicacid sequence A to, with, or against a given nucleic acid sequence B(which can alternatively be phrased as a given nucleic acid sequence Athat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence B) is calculated asfollows:

-   -   100 times the fraction X/Y        where X is the number of nucleic acid residues scored as        identical matches by the sequence in that program's alignment of        A and B, and where Y is the total number of nucleic acid        residues in B. It will be appreciated that where the length of        nucleic acid sequence A is not equal to the length of nucleic        acid sequence B, the % nucleic acid sequence identity of A to B        will not equal the % nucleic acid sequence identity of B to A.

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. In one embodiment, the disorder or disease is cancer. Inanother embodiment, the disorder or disease is metastatic cancer.

The term “cancer” refers to or describes the physiological condition inmammals that is typically characterized by unregulated cellgrowth/proliferation. Examples of cancer include hut are not limited to,lymphoma, blastoma, sarcoma, and leukemia.

The terms “metastatic cancer” or “cancer metastasis” refer to a primarycancer capable of metastasis or cancer which has spread (i.e.,metastasized) from a primary cancer, primary cancerous tissue or primarycancerous cells (e.g., cancer stem cells) from one part of of the bodyto one or more other parts of the body to form a secondary cancer orsecondary cancers. Metastatic cancer or cancer metastasis also refer tolocally advanced cancer that has spread from a primary cancer to nearbytissue(s) or lymph node(s). Metastatic cancer includes tumors that aredefined as being high grade and/or high stage, for example tumors with aGleason score of 6 or higher in prostate cancer are more likely tometastasize, Metastatic cancer also refers to tumors defined by one ormore molecular markers that correlate with the metastasis.

The terms “relapsed cancer”, “relapse of a cancer”, “cancer relapse”, or“tumor relapse” refer to the return or reappearance of cancer after aperiod of improvement. Typically the period of improvement is afteradministration of a therapy that resulted in the decrease of ordisappearance of signs and symptoms of cancer. The period of improvementcan be the decrease or disappearance of all signs and symptoms ofcancer. The period of improvement can also be the decrease ordisappearance of some, but not all, signs and symptoms of cancer. Insome embodiments, the relapsed cancer is a cancer that has becomeunresponsive or partially unresponsive to a drug or a therapy. Forexample and without limitation, relapsed cancer includes cancer inpatients whose first progression occurs in the absence of any treatmentfollowing successful treatment with a drug or a therapy; cancer inpatients who progress on a treatment, or within 60 days of thetreatment; and cancer in patients who progress while receivingtreatment.

The terms “cancer stem cell”, “cancer stem cells” or “CSCs” as usedherein refer to a subpopulation of tumor cells or cancer cells. Cancerstem cells possess characteristics associated with normal stem cells,such as the ability to give rise to different cell types found in aparticular cancer or tumor. Cancer stem cells have the capacity to drivethe production or formation of a tumor or tumors through self-renewaland/or differentiation. Cancer stem cells have been identified in anumber of cancers including, but not limited to, breast, brain, blood,liver, kidney, cervical, ovarian, colon, and lung cancers among others.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing or suppressing metastasis, decreasing the rate ofdisease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments,oligonucleotides described herein are used to prevent or suppressmetastasis. An individual is successfully “treated”, for example, usingan oligonucleotide of the invention if the individual shows observableand/or measurable reduction in or absence of one or more of thefollowing: reduction in the number of cancer cells or absence of thecancer cells; reduction in the tumor size; inhibition (i.e., slow tosome extent and preferably stop) of cancer cell infiltration intoperipheral organs including the spread of cancer into soft tissue andbone; inhibition (i.e., slow to some extent and preferably stop) oftumor metastasis; inhibition, to some extent, of tumor growth or tumorrelapse; and/or relief to some extent, one or more of the symptomsassociated with the specific cancer; reduced morbidity and mortality,and improvement in quality of life issues.

As used herein, the term “prevention” includes providing prophylaxiswith respect to occurrence or recurrence of a disease in an individual.An individual may be predisposed to, susceptible to a disorder, or atrisk of developing a disorder, but has not yet been diagnosed with thedisorder. In some embodiments, oligonucleotides described herein areused to prevent or suppress metastasis.

As used herein, an individual “at risk” of developing a disorder may ormay not have detectable disease or symptoms of disease, and may or maynot have displayed detectable disease or symptoms of disease prior tothe treatment methods described herein. “At risk” denotes that anindividual has one or more risk factors, which are measurable parametersthat correlate with development of cancer (e.g., metastatic cancer), asknown in the art. An individual having one or more of these risk factorshas a higher probability of developing the disorder than an individualwithout one or more of these risk factors.

An “individual” or “subject” can be a vertebrate, a mammal, or a human.Mammals include, but are not limited to, farm animals (such as cows),sport animals, pets (such as horses), primates, mice and rats.Individuals also include companion animals including, but not limitedto, dogs and cats. In one aspect, an individual is a human.

A “healthcare professional,” as used herein, can include, withoutlimitation, doctors, nurses, physician assistants, lab technicians,research scientists, clerical workers employed by the same, or anyperson involved in determining, diagnosing, aiding in the diagnosis orinfluencing the course of treatment for the individual.

An “effective amount” refers to an amount of therapeutic compound, suchas an oligonucleotide or other anticancer therapy, administered to anindividual, either as a single dose or as part of a series of doses,which is effective to produce a desired therapeutic or prophylacticresult.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disorder. A therapeutically effective amount herein may varyaccording to factors such as the disease state, age, sex, and weight ofthe patient, and the ability of the oligonucleotide to elicit a desiredresponse in the individual. A therapeutically effective amount may alsobe one in which any toxic or detrimental effects of the oligonucleotideare outweighed by the therapeutically beneficial effects. In the case ofcancer, the therapeutically effective amount of the oligonucleotide mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the oligonucleotide may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic.

A “prophylactically effective amount” refers to an amount effective, atthe dosages and for periods of time necessary, to achieve the desiredprophylactic result. For example, a prophylactically effective amount ofthe oligonucleotides of the present invention is at least the minimumconcentration that prevents or attenuates the development of at leastone symptom of metastatic cancer.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “pharmaceutical formulation” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective, and that contains no additional components that areunacceptably toxic to a subject to which the formulation would beadministered. Such formulations are sterile.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

III. Oligonucleotides and Other Anti-Cancer Therapies

Human cells express a number of unique chimeric mitochondrial RNAmolecules. These molecules are non-coding (i.e., they are not known toserve as a template for the translation of a protein) and comprise the16S mitochondrial ribosomal RNA covalently linked at the 5′ end to aninverted repeat sequence. Chimeric mitochondrial RNA molecules are foundin two forms: sense and antisense.

The sense chimeric non-coding mitochondrial RNA (SncmtRNA) moleculecorresponds to the 16S mitochondria(ribosomal RNA transcribed from the“H-strand” of the circular mitochondrial genome. Covalently linked tothe 5′ end of this RNA molecule is a nucleotide sequence or invertedrepeat sequence corresponding to an RNA transcribed from the “L-strand”of the mitochondrial 16S gene. The size of the inverted repeat sequencein the SncmtRNA can vary from about 25, 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 nucleotides or moreto between about 100-200, 1150-250, 200-300, 250-350, 400-500, 450-550,500-600, 550-650, 600-700, 650-750, or 700-800 nucleotides or more,including any number in between these values. In one embodiment, theinverted repeat sequence in the SncmtRNA corresponds to a fragment of815 nucleotides of the RNA transcribed from the L-strand of the 16S geneof the mitochondrial genome. In another embodiment, the inverted repeatsequence in the SncmtRNA corresponds to a fragment of 754 nucleotides ofthe RNA transcribed from the L-strand of the 16S gene of themitochondrial genome. In still another embodiment, the inverted repeatsequence in the SncmtRNA corresponds to a fragment of 694 nucleotides ofthe RNA transcribed from the L-strand of the 16S gene of themitochondrial genome. In another embodiment, the SncmtRNA corresponds toSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In another embodiment, theSncmtRNA comprises a sequence selected from the group consisting of SEQID NO:200, SEQ ID NO:201, and SEQ ID NO:202.

The antisense chimeric non-coding mitochondrial RNA (ASncmtRNA) moleculecorresponds to the 16S mitochondrial ribosomal RNA transcribed from the“L-strand” of the circular mitochondrial genome. Covalently linked tothe 5′ end of this RNA molecule is a nucleotide sequence or the invertedrepeat sequence corresponding to an RNA transcribed from the “H-strand”of the mitochondrial 16S gene. The size of the inverted repeat sequencein the ASncmtRNA can vary from about 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800 nucleotides ormore to between about 100-200, 150-250, 200-300, 250-350, 400-500,450-550, 500-600, 550-650, 600-700, 650-750, or 700-800 or more,including any number in between these values. In another embodiment, theASncmtRNA corresponds to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. Inanother embodiment, the ASncmtRNA comprises a sequence selected from thegroup consisting of SEQ ID NO:203, SEQ ID NO:204, and SEQ ID NO:205.

Further information related to chimeric mitochondrial RNA molecules canbe found in U.S. Pat. No. 8,318,686, the disclosure of which isincorporated by reference herein in its entirety.

In one aspect, the invention provides one or more oligonucleotidecomplementary to an ASncmtRNA molecule or a SncmtRNA molecule, whereinthe oligonucleotide is able to hybridize with the chimeric mitochondrialRNA molecules to form a stable duplex for use in a method disclosedherein. In some aspects, provided herein are methods for suppressingmetastasis of a cancer in an individual using one or moreoligonucleotides described herein. In some aspects, provided herein aremethods for treating or preventing relapse of a cancer in an individual.In some aspects, provided herein are methods for treating metastaticcancer in an individual. In some embodiments herein, the individual hasbeen previously treated for cancer with a therapy (e.g., chemotherapy,radiation therapy, surgery or combinations thereof). In someembodiments, the one or more oligonucleotide complementary to anASncmtRNA molecule or a SncmtRNA molecule described herein has or moreof the following characteristics when used in a method disclosed herein:(1) hybridizes with the chimeric mitochondrial RNA molecules (i.e., anASncmtRNA molecule or a SncmtRNA molecule) to form a stable duplex; (2)hybridizes with the chimeric mitochondrial RNA molecules expressed bytumor cells and inhibits, arrests, kills or abolishes tumor cells; (3)hybridizes with the chimeric mitochondrial RNA molecules expressed bycancer stem cells (CSCs) and inhibits, arrests, kills or abolishes CSCs;(4) suppresses metastasis of a cancer in an individual (e.g., anindividual previously treated for cancer with a therapy); (5) treats orprevents relapse of a cancer in an individual (e.g., an individualpreviously treated for cancer with a therapy); (6) treats metastaticcancer in an individual (e.g., an individual previously treated forcancer with a therapy); and (7) prolongs overall survival in anindividual previously treated for cancer with a therapy (e.g.,chemotherapy, radiation therapy, surgery or combinations thereof).

In one aspect, the oligonucleotides for use in any of the methodsdescribed herein can be complementary to a SncmtRNA molecule and/or toan ASncmtRNA molecule disclosed herein. Without being bound to theory,it is believed that the complementary oligonucleotides bind to thencmtRNAs and interfere with their cellular functions. As used herein, anoligonucleotide sequence is “complementary” to a portion of an ncmtRNA,as referred to herein, if the oligonucleotide possesses a sequencehaving sufficient complementarity to be able to hybridize with thencmtRNA to form a stable duplex. The ability to hybridize will depend onboth the degree of complementarity and the length of theoligonucleotide. Generally, the longer the hybridizing oligonucleotide,the more base mismatches with an ncmtRNA it may contain and still form astable duplex. In some aspects, the one or more oligonucleotide usedaccording to the methods disclosed herein is at least 8 (such as atleast 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 or more) base pairs in length. Thoseskilled in the art can ascertain a tolerable degree of mismatch by useof standard procedures to determine the melting point of the hybridizedcomplex. In some embodiments, the one or more oligonucleotide is atleast 85% (such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) complementary to a SncmtRNA moleculeand/or to a ASncmtRNA molecule disclosed herein. In some embodiments,the complementary oligonucleotide is an antisense oligonucleotide. Inone embodiment, the one or more oligonucleotide is complementary to oneor more ncmtRNA encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:1-6. In some embodiments, the one or moreoligonucleotide comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:7-198. In some embodiments, the one ormore oligonucleotide comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:36, 197 and 198. In some embodiments, theone or more oligonucleotide comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs:36, 197 and 198.

a. Oligonucleotide Modifications

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5 phosphodiester linkage. The oligonucleotides (e.g., an antisenseoligonucleotide) used for suppressing metastasis of a cancer, treatingor preventing relapse of a cancer, or treating metastatic canceraccording to any of the methods disclosed herein can have one or moremodified, i.e. non-naturally occurring, internucleoside linkages. Withrespect to therapeutics, modified internucleoside linkages are oftenselected over oligonucleotides having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases present inbodily fluids.

Oligonucleotides (e.g., an antisense oligonucleotide) having modifiedinternucleoside linkages include internucleoside linkages that retain aphosphorus atom as well as internucleoside linkages that do not have aphosphorus atom. Representative phosphorus containing internucleosidelinkages include, but are not limited to, phosphodiesters,phosphotriesters, methylphosphonates, phosphoramidate, andphosphorothioates. Methods of preparation of phosphorous-containing andnon-phosphorous-containing linkages are well known in the art.

In one embodiment, oligonucleotides (e.g., an antisense oligonucleotide)targeted to a SncmtRNA molecule and/or to an ASncmtRNA moleculedisclosed herein comprise one or more modified internucleoside linkages.In some embodiments, the modified internucleoside linkages arephosphorothioate linkages.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific, though nonlimiting, examples of oligonucleotides (e.g., anantisense oligonucleotide) useful in the methods of the presentinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

In some embodiments, modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thiono-phosphoramidates,thionoalkylphosphonates, thionoalkylphospho-triesters, selenophosphatesand boranophosphates having normal linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity comprise, a single 3′ to 3′linkage at the 3′-most internucleotide, linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof) can also be employed. Various salts,mixed salts and free acid forms are also included. Oligonucleotidebackbones that do not include, a phosphorus atom therein have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatamic or heterocyclicinternucleoside linkages. These include those having morpholino linkages(formed in part from the sugar portion of a nucleoside); siloxanebackbones; sulfide, sulfoxide and sulfone backbones; formacetyl andthioformacetyl backbones; methylene formacetyl and thioformacetylbackbones; riboacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

In other embodiments, both the sugar and the internucleoside linkage,i.e., the backbone, of the nucleotide units are replaced with novelgroups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,an oligonucleotide mimetic is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 254:14974500, 1991.

Representative United States patents that teach the preparation of theabove phosphorus-containing and non-phosphorus-containing linkagesinclude, but are not limited to, U.S. Pat. Nos. 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;5,721,218; 5,672,697 and 5,625,050, 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

Modified oligonucleotides (e.g., antisense oligonucleotides)complementary to SncmtRNA and/or ASncmtRNA used as anticancer therapiesin combination with any of the methods disclosed herein (e.g., method ofsuppressing metastasis of a cancer) may also contain one or moresubstituted sugar moieties. For example, the furanosyl sugar ring can bemodified in a number of ways including substitution with a substituentgroup, bridging to form a bicyclic nucleic acid “BNA” and substitutionof the 4′-O with a heteroatom such as S or N(R) as described in U.S.Pat. No. 7,399,845, hereby incorporated by reference herein in itsentirety. Other examples of BNAs are described in publishedinternational Patent Application No. WO 2007/146511, hereby incorporatedby reference herein in its entirety.

The oligonucleotides (e.g., antisense oligonucleotides) for use in themethods disclosed herein (e.g., method of suppressing metastasis of acancer) can optionally contain one or more nucleotides having modifiedsugar moieties. Sugar modifications may impart nuclease stability,binding affinity or some other beneficial biological property to theantisense compounds. The furanosyl sugar ring of a nucleoside can bemodified in a number of ways including, but not limited to: addition ofa substituent group, particularly at the 2′ position; bridging of twonon-geminal ring atoms to form a bicyclic nucleic acid (BNA); andsubstitution of an atom or group such as —S—, —N(R)— or —C(R1)(R2) forthe ring oxygen at the 4′-position. Modified sugars include, but are notlimited to: substituted sugars, especially 2′-substituted sugars havinga 2′-F, 2′-OCH2 (2′-OMe) or a 2″-O (CH₂)₂—OCH₃ (2′-O-methoxyethyl or2′-MOE) substituent group; and bicyclic modified sugars (BNAs), having a4′-(CH₂)n-O-2″ bridge, where n=1 or n=2. Methods for the preparations ofmodified sugars are well known to those skilled in the art.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In other embodiments, the bicyclic sugar moiety comprises a bridge groupbetween the 2′ and the 4′-carbon atoms. In certain such embodiments, thebridge group comprises from 1 to linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises from 1 to 4 linkedbiradical groups. In certain embodiments, the bicyclic sugar moietycomprises 2 or 3 linked biradical groups. In certain embodiments, thebicyclic sugar moiety comprises 2 linked biradical groups. In certainembodiments, a linked biradical group is selected from —O—, —S—,—N(R1)-, —C(R1)(R2)-, —C(R1)═C(R1)-, —C(R1)═N—, —C(═NR1)—, —Si(R1)(R2)-,—S(═O)2—, —S(═O)—, —C(═O)— and —C(═S)—; where each R1 and R2 is,independently, H, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl,C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substitutedC2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycleradical, a substituted hetero-cycle radical, heteroaryl, substitutedheteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclicradical, halogen, substituted oxy (—O—), amino, substituted amino,azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN,thiol, substituted thiol, sulfonyl (S(═O)2—H), substituted sulfonyl,sulfoxyl (S(═O)—H) or substituted sulfoxyl; and each substituent groupis, independently, halogen, C1-C12 alkyl, substituted C1-C12 alkyl,C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substitutedC2-C12 alkenyl, amino, substituted amino, acyl, substituted acyl, C1-C12aminoalkyl, C1-C12 aminoalkoxy, substituted C1-C12 aminoalkyl,substituted C1-C12 aminoalkoxy or a protecting group.

Oligonucleotides (e.g., antisense oligonucleotides) for use in any ofthe methods disclosed herein (e.g., method of suppressing metastasis ofa cancer) may also include nucleobase (often referred to in the artsimply as “base”) modifications or substitutions. Nucleobasemodifications or substitutions are structurally distinguishable from,yet functionally interchangeable with, naturally occurring or syntheticunmodified nucleobases. Both natural and modified nucleobases arecapable of participating in hydrogen bonding. Such nucleobasemodifications may impart nuclease stability, binding affinity or someother beneficial biological property to oligonucleotide compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly ⁻useful for increasing the binding affinity of anoligonucleotide compound (such as an antisense oligonucleotide compound)for a target nucleic acid (such as an ncmtRNA).

Additional unmodified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U).

Modified nucleobases include other synthetic and natural nucleobasessuch as 5-methylcytosine (5-me-C), 5-hydroxyrnethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), O-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(1-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302 Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, U.S. Pat. Nos.5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; and 5,681,941, each of which is herein incorporated byreference.

b. Ribozymes

In another embodiment of the invention, ribozymes can be used tointerfere with the ncmtRNA molecules described herein to induce celldeath in proliferative cells associated with mestastasis (e.g., CSCs).The sequence of the ribozyme can be designed according to the sequenceof the ASncmtRNA (for example, a sequence corresponding to SEQ ID NO:4,SEQ ID NO:5, or SEQ ID NO:6, or a sequence comprising SEQ ID NO:203, SEQID NO:204, or SEQ ID NO:205) or the SncmtRNA (for example, a sequencecorresponding to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a sequencecomprising SEQ ID NO:200, SEQ ID NO:202, or SEQ ID NO:203) to cleavespecific regions of the transcript. Ribozymes are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA (Rossi,Curr. Biology 4:469-471, 1994). The mechanism of ribozyme actioninvolves sequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage. Thecomposition of ribozyme molecules must include one or more sequencescomplementary to the RNA, and must include the well-known catalyticsequence responsible for RNA cleavage, and described in U.S. Pat. No.5,093,246, the disclosure of which is incorporated by reference hereinin its entirety. As such, within the scope of the invention, hammerheadribozyme molecules can be engineered that specifically and efficientlycatalyze endonucleolytic cleavage of the ASncmtRNA or SncmtRNA moleculesdisclosed herein. The construction and production of hammerheadribozymes is well known in the art and it was described (Haseloff etal., Gene, 82:43-52, 1989). Ribozymes of the present invention can alsoinclude RNA endoribonucleases (Zaug et al., Science, 224:574-578, 1984).In some embodiments, a ribozyme described herein can be used in anymethod described herein. In some embodiments, a method of suppressingmetastasis of a cancer in an individual comprises administering to theindividual an effective amount of one or more ribozyme described herein.In some embodiments, a method for treating or preventing relapse ofcancer in an individual comprises administering to the individual aneffective amount of one or more ribozyme described herein. In someembodiments, a method for treating metastatic cancer in an individualcomprises administering to the individual an effective amount of one ormore ribozyme described herein. In some embodiments, a method fortreating a refractory cancer (e.g., a refractory HPV-associated cancer)in an individual comprises administering to the individual an effectiveamount of one or more ribozyme described herein. In some embodiments,the individual has been previously treated for cancer with a therapy(e.g., chemotherapy, radiation therapy, surgery or combinationsthereof).

c. RNA Interference

In another aspect, interference with the function of the ASncmtRNAand/or SncmtRNA molecules disclosed herein for use in any of the methodsdisclosed herein (e.g., method of suppressing metastasis of a cancer)can be achieved by RNA interference or RNA silencing. RNA interference(RNAi) has emerged as a novel and promising approach for gene silencingin mammalian cells (Elbashir et al., Nature 411:494-498, 2001; McManuset al., Nature Rev. Genet, 3:737-747, 2002). Synthetically synthesizeddouble stranded RNA molecules of about 8 to 40 (such as about 10 to 36,14 to 32, 18-28, or 22-24) base pairs (bp) or at least about 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp in length hybridizespecifically to their complementary target RNA, leading to degradationof the RNA. Several different genes have been silenced successfully bysmall interfering RNA or siRNA (Lu et al., Curr. Opin. Mol. Ther.5:225-234, 2003; Wacheck et al., Oligonucleotides 13:393-400, 2003).Therefore, synthetic double stranded RNA targeted to the ASncmtRNAand/or SncmtRNA molecules disclosed herein can be used to degrade thesetranscripts and induce cancer cell death (e.g., CSC death). Thosefamiliar in the art will understand that the sequence of the siRNA hasto be complementary to any region of the ASncmtRNA and/or SncmtRNAmolecules (such as complementary to any one of a sequence correspondingto SEQ NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and/orSEQ ID NO:6, or complementary to any one of a sequence comprising SEQ IDNO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204,and/or SEQ ID NO:205). In some embodiments, an RNA described herein canbe used in any method described herein. In some embodiments, a method ofsuppressing metastasis of a cancer in an individual comprisesadministering to the individual an effective amount of one or more RNAdescribed herein. In some embodiments, a method for treating orpreventing relapse of cancer in an individual comprises administering tothe individual an effective amount of one or more RNA described herein.In sonic embodiments, a method for treating metastatic cancer in anindividual comprises administering to the individual an effective amountof one or more RNA described herein. In some embodiments, a method fortreating a refractory cancer (e.g., a refractory HPV-associated cancer)in an individual comprises administering to the individual an effectiveamount of one or more RNA described herein. In some embodiments, theindividual has been previously treated for cancer with a therapy (e.g.,chemotherapy, radiation therapy, surgery or combinations thereof).

d. Oligonucleotide Delivery

In one embodiment, a recombinant vector can be used for delivering oneor more oligonucleotides (such as any of the oligonucleotides disclosedherein) complementary to a sense and/or antisense chimeric non-codingmitochondrial RNA molecule to the individual. This can include bothsystemic delivery and delivery localized to a particular region of thebody (such as, the bone marrow). Any vector capable of enablingrecombinant production of one or more oligonucleotides complementary toa sense or antisense chimeric ncmtRNA molecule and/or which can deliverone or more oligonucleotides complementary to a sense or antisensechimeric ncmtRNA molecule into a host cell is contemplated herein. Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid. The vector can be part of a DNAvaccine or used as part of any other method for delivering aheterologous gene for expression in a host cell that is known to onehaving skill in the art. Recombinant vectors are capable of replicatingwhen transformed into a suitable host cell. Viral vectors infect a widerange of non-dividing human cells and have been used extensively in livevaccines without adverse side effects. A viral vector (such as, but notlimited to, an adenoviral vector or an adeno-associated viral (AAV)vector (e.g. AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. or hybridAAV vectors comprising the same) is an example of a vector for use inthe present methods for delivering one or more oligonucleotidescomplementary to a sense or antisense chimeric ncmtRNA molecule tocancer cells (such as a plasmocyte; see, e.g. U.S. Patent ApplicationPublication No. 2004/0224389, the disclosure of which is incorporated byreference herein, or a CSC). In some embodiments, a recombinant vector(e.g., a viral vector) described herein can be used in any methoddescribed herein. In some embodiments, a method of suppressingmetastasis of a cancer in an individual comprises administering to theindividual an effective amount of a recombinant vector (e.g., a viralvector) comprising one or more oligonucleotide described herein. In someembodiments, a method for treating or preventing relapse of cancer in anindividual comprises administering to the individual an effective amountof a recombinant vector (e.g., a viral vector) comprising one or moreoligonucleotide described herein. In some embodiments, a method fortreating metastatic cancer in an individual comprises administering tothe individual an effective amount of a recombinant vector (e.g., aviral vector) comprising one or more oligonucleotide described herein.In some embodiments, a method for treating a refractory cancer (e.g., arefractory HPV-associated cancer) in an individual comprisesadministering to the individual an effective amount of a recombinantvector (e.g., a viral vector) comprising one or more oligonucleotidedescribed herein. In some embodiments, the individual has beenpreviously treated for cancer with a therapy (e.g., chemotherapy,radiation therapy, surgery or combinations thereof).

In another aspect, one or more oligonucleotides (such as any of theoligonucleotides disclosed herein) complementary to a sense and/orantisense chimeric non-coding mitochondrial RNA molecule is encapsulatedwithin a microcarrier for deliver to an individual. In certainembodiments, a mixture of different oligonucleotides (such as any of theoligonucleotides disclosed herein) complementary to a sense and/orantisense chimeric non-coding mitochondrial RNA molecule may beencapsulated with a microcarrier, such that the microcarrierencapsulates more than one oligonucleotide species. In some embodiments,the one or more oligonucleotides (such as any of the oligonucleotidesdisclosed herein) complementary to a sense and/or antisense chimericnon-coding mitochondrial RNA molecule encapsulated within themicrocarrier comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:7-198. In some embodiments, the one or moreoligonucleotides (such as any of the oligonucleotides disclosed herein)complementary to a sense and/or antisense chimeric non-codingmitochondrial RNA molecule encapsulated within the microcarriercomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:36, 197 and 198.

Methods of encapsulating oligonucleotides in microcarriers are wellknown in the art, and described, for example, International applicationWO98/55495. Colloidal dispersion systems, such as microspheres, beads,macromolecular complexes, nanocapsules and lipid-based system, such asoil-in-water emulsions, micelles, mixed micelles and liposomes canprovide effective encapsulation of oligonocelotides within microcarriercompositions. The encapsulation composition may further comprise any ofa wide variety of components. These include, but are not limited to,alum, lipids, phospholipids, lipid membrane structures (LMS),polyethylene glycol (PEG) and other polymers, such as polypeptides,glycopeptides, and polysaccharides.

Other Anti-Cancer Therapies

In some aspects, any of the methods of treatment described herein cancomprise administering one or more additional anti-cancer therapies tothe individual. In some embodiments, the one or more anti-cancer therapyis selected from the group consisting of chemotherapy, radiationtherapy, and surgery. Chemotherapy and anti-cancer agents are usedinterchangeably herein. Various classes of anti-cancer agents can beused. Non-limiting examples include: alkylating agents, antimetabolites,anthracyclines, plant alkaloids, topoisomerase inhibitors,podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), tyrosinekinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)),hormone treatments, soluble receptors and other antineoplastics.

Topoisomerase inhibitors are also another class of anti-cancer agentsthat can be used herein. Topoisomerases are essential enzymes thatmaintain the topology of DNA. Inhibition of type I or type IItopoisomerases interferes with both transcription and replication of DNAby upsetting proper DNA supercoiling. Some type I topoisomeraseinhibitors include camptothecins: irinotecan and topotecan. Examples oftype II inhibitors include amsacrine, etoposide, etoposide phosphate,and teniposide. These are semisynthetic derivatives ofepipodophyllotoxins, alkaloids naturally occurring in the root ofAmerican Mayapple (Podophyllum peltatum).

Antineoplastics include the immunosuppressant dactinomycin, doxorubicin,epirubicin, bleomycin, mechlorethamine, cyclophosphamide, chlorambucil,ifosfamide. The antineoplastic compounds generally work by chemicallymodifying a cell's DNA.

Alkylating agents can alkylate many nucleophilic functional groups underconditions present in cells. Cisplatin and carboplatin, and oxaliplatinare alkylating agents. They impair cell function by forming covalentbonds with the amino, carboxyl, sulfhydryl, and phosphate groups inbiologically important molecules.

Vinca alkaloids bind to specific sites on tubulin, inhibiting theassembly of tubulin into microtubules (VI phase of the cell cycle). Thevinca alkaloids include: vincristine, vinblastine, vinorelbine, andvindesine.

Anti-metabolites resemble purines (azathioprine, mercaptopurine) orpyrimidine and prevent these substances from becoming incorporated in toDNA during the “S” phase of the cell cycle, stopping normal developmentand division. Anti-metabolites also affect RNA synthesis.

Plant alkaloids and terpenoids are derived from plants and block celldivision by preventing microtubule function. Since microtubules arevital liar cell division, without them, cell division cannot occur. Themain examples are vinca alkaloids and taxanes.

Podophyllotoxin is a plant-derived compound which has been reported tohelp with digestion as well as used to produce two other cytostaticdrugs, etoposide and teniposide. They prevent the cell from entering theG1 phase (the start of DNA replication) and the replication of DNA (theS phase).

Taxanes as a group includes paclitaxel and docetaxel. Paclitaxel is anatural product, originally known as Taxol and first derived front thebark of the Pacific Yew tree. Docetaxel is a semi-synthetic analogue ofpaclitaxel. Taxanes enhance stability of microtubules, preventing theseparation of chromosomes during anaphase.

In some aspects, the anti-cancer agent can be selected from remicade,docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids,gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide,temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan,methotrexate, gefitinib (Iressa®), taxol, taxotere, fluorouracil,leucovorin, irinotecan, xeloda, CPT-11, interferon alpha, pegylatedinterferon alpha (e.g., PEG INTRON-A), capecitabine, cisplatin,thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine,doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine,vinorelbine, zoledronic acid, palmitronate, biaxin, busulphan,prednisone, bortezomib (Velcade®), bisphosphonate, arsenic trioxide,vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin,estrainustine sodium phosphate (Emcyt®), sulindac, or etoposide.

In other embodiments, the anti-cancer agent can be selected frombortezomib, cyclophosphamide, dexamethasone, doxorubicin,interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha,prednisone, thalidomide, or vincristine.

In some aspects, the one or more anti-cancer therapy is radiationtherapy. As used herein, the term “radiation therapy” refers to theadministration of radiation to kill cancerous cells. Radiation interactswith molecules in the cell such as DNA to induce cell death. Radiationcan also damage the cellular and nuclear membranes and other organelles.Depending on the radiation type, the mechanism of DNA damage may vary asdoes the relative biologic effectiveness. For example, heavy particles(i.e. protons, neutrons) damage DNA directly and have a greater relativebiologic effectiveness. Electromagnetic radiation results in indirectionization acting through short-lived, hydroxyl free radicals producedprimarily by the ionization of cellular water. Clinical applications ofradiation consist of external beam radiation (from an outside source)and brachytherapy (using a source of radiation implanted or insertedinto the patient). External beam radiation consists of X-rays and/orgamma rays, while brachytherapy employs radioactive nuclei that decayand emit alpha particles, or beta particles along with a gamma ray.Radiation also contemplated herein includes, for example, the directeddelivery of radioisotopes to cancer cells. Other forms of DNA damagingfactors are also contemplated herein such as microwaves and UVirradiation.

Radiation may be given in a single dose or in a series of small doses ina dose-fractionated schedule. The amount of radiation contemplatedherein ranges from about 1 to about 100 Gy, including, for example,about 5 to about 80, about 10 to about 50 Gy, or about 10 Gy. The totaldose may be applied in a fractioned regime. For example, the regime maycomprise fractionated individual doses of 2 Gy. Dosage ranges forradioisotopes vary widely, and depends on the half-life of the isotopeand the strength and type of radiation emitted, When the radiationcomprises use of radioactive isotopes, the isotope may be conjugated toa targeting agent, such as a therapeutic antibody, which carries theradionucleotide to the target tissue (e.g., tumor tissue). Suitableradioactive isotopes include, but are not limited to, astatine²¹¹,¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ^(s7)iron, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu,gallium⁶⁷, ̂hydrogen, iodine¹²³, iodine¹³¹, indium¹¹¹, ⁵⁹ion,³²phosphorus, rhenium¹⁸⁶, ⁷⁵selenium, ³⁵sulphur, technicium^(99m),and/or yttrium^(o).

Surgery described herein includes resection in which all or part of acancerous tissue is physically removed, exercised, and/or destroyed.Tumor resection refers to physical removal of at least part of a tumor.In addition to tumor resection, treatment by surgery includes lasersurgery, cryosurgery, electrosurgery, and micropically controlledsurgery (Mohs surgery). Removal of precancers or normal tissues is alsocontemplated herein.

Stem Cell Transplantation and Ex Vivo Treatment of AutologousHematopoietic Stem Cells

In other aspects, any of the methods of treatment described herein caninclude either autologous or allogenic stem cell transplantationtherapy. In recent years, high-dose chemotherapy with autologoushematopoietic stem-cell transplantation has become the preferredtreatment for certain cancers such as multiple myeloma, non-Hodgkinlymphoma, Hodgkin lymphoma, and leukemia. While not curative, thisprocedure does prolong overall survival and complete remission. Prior tostem-cell transplantation, patients receive an initial course ofinduction chemotherapy. The most common induction regimens used todayare thalidomide-dexamethasone, hortezornib based regimens, andlenalidomide-dexamethasone (Kyle & Rajkumar, Blood, 111 (6): 2962-72,2008). For example, autologous peripheral stem cell transplantation isuseful for up to 50% of multiple myeloma patients. Despite a lowmortality rate, problems with such transplant therapy include theinability to eradicate the tumor and the difficulty in the removal ofmyeloma cells and their precursors from the stem cell collection usedfor transplantation.

Allogenic transplant (the transplantation of a healthy person's stemcells into the affected individual), is another therapy option fortreating certain cancers such as multiple myeloma, non-Hodgkin lymphoma,Hodgkin lymphoma, and leukemia but is less frequently used as it may notprovide a cure. For example, most studies evaluating its use in multiplemyeloma patients demonstrate long-term disease-free survival of 10-20%,with a significant fraction of patients developing relapse.

When included as a treatment for suppressing or preventing metastasisaccording to any of the methods disclosed herein, autologous stem celltransplantation can also include the step of treating the hematopoieticstem-cells and/or bone marrow to he transplanted into the affectedindividual with any of the anti-cancer agents disclosed herein, prior totransplantation into the affected individual. In one embodiment,hematopoietic stem-cells and/or bone marrow for use in autologous stemcell transplantation can be treated with an effective amount of one ormore oligonucleotides (e.g., antisense oligonucleotides) sufficientlycomplementary to an ASncmtRNA or SncmtRNA molecule (e.g., any of theASncmtRNA and/or SncmtRNA molecules disclosed herein) to form a stableduplex prior to transplantation into the affected individual. In anotherembodiment, the one or more oligonucleotide is sufficientlycomplementary to one or more ncmtRNA encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:1-6, to form a stableduplex. In other embodiments, the one or more oligonucleotide comprisesa nucleic acid sequence selected from the group consisting of SEQNOs:7-198. In some embodiments, the one or more oligonucleotidecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:36, 197 and 198.

It has been shown that autologous transplantation of bone marrow orhematological stem cells can also be used to treat several forms ofhematological cancers (such as, but not limited to, multiple myeloma,leukemia and lymphoma). Accordingly, in some aspects, when included as atreatment for a hematological cancer, provided herein is a method ofperforming autologous stem cell transplantation which includes the stepof treating the hematopoietic stern-cells and/or bone marrow to betransplanted into the affected individual with any of the anti-canceragent disclosed herein, prior to transplantation into the affectedindividual. In one embodiment, hematopoietic stem-cells and/or bonemarrow for use in autologous stem cell transplantation in an individualwith a hematological cancer can be treated with an effective amount ofone or more oligonucleotides (e.g., antisense oligonucleotides)sufficiently complementary to an ASncmtRNA molecule or SncmtRNA molecule(e.g., any of the ASncmtRNA and/or SncmtRNA molecules disclosed herein)to form a stable duplex prior to transplantation into the affectedindividual. In another embodiment, the one or more oligonucleotide issufficiently complementary to one or more ncmtRNA encoded by a nucleicacid sequence selected from the group consisting of SEQ ID NOs:1-6, toform a stable duplex. In other embodiments, the one or moreoligonucleotide comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:7-198. In some embodiments, the one ormore oligonucleotide comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:36, 197 and 198.

IV. Compositions

Any of the anti-cancer agents (such as oligonucleotide-based agents)disclosed herein can be administered in the form of compositions (e.g.,pharmaceutical compositions). These compounds can be administered bysystemic administration or local administration through various routes.The route(s) of administration useful in a particular application areapparent to one of skill in the art. Routes of administration includebut are not limited to oral, rectal, cerebrospinal, transdermal,subcutaneous, topical, transmucosal, nasopharangeal, pulmonary,intravenous, intramuscular, and intranasal. In some embodiments, theadministration is a local administration. In some embodiments, the localadministration is selected from the group consisting of administrationinto an organ, into a cavity, into a tissue, and subcutaneousadministration. In some embodiments, the administration is systemicadministration. In some embodiments, the systemic administration isintravenous or intraperitoneal administration. These compounds areeffective as both injectable and oral compositions. Such compositionsare prepared in a manner well known in the pharmaceutical art andcomprise at least one active compound. The compositions herein may alsocontain more than once active compound as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. When employed as oralcompositions, the oligonucleotides, and other anti-cancer agentsdisclosed herein, are protected from acid digestion in the stomach by apharmaceutically acceptable protectant.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the anti-cancer agentsdisclosed herein associated with one or more pharmaceutically acceptableexcipients or carriers. In making the compositions of this invention,the active ingredient is usually mixed with an excipient or carrier,diluted by an excipient or carrier or enclosed within such an excipientor carrier which can be in the form of a capsule, sachet, paper or othercontainer. When the excipient or carrier serves as a diluent, it can bea solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

In some embodiments, in preparing a formulation, it may be necessary tomill the active lyophilized compound to provide the appropriate particlesize prior to combining with the other ingredients. If the activecompound is substantially insoluble, it ordinarily is milled to aparticle size of less than 200 mesh. the active compound issubstantially water soluble, the particle size is normally adjusted bymilling to provide a substantially uniform distribution in theformulation, e.g. about 40 mesh.

Some examples of suitable excipients or carriers include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterilewater, syrup, and methyl cellulose. The formulations can additionallyinclude: lubricating agents such as talc, magnesium stearate, andmineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. The compositions of theinvention can be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after administration to the patient byemploying procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 mg to about 100 mg or more, such as any one ofabout 1 mg, to about 5 mg, I1 mg to about 10 mg, about 1 mg to about 20mg, about 1 mg to about 30 mg, about 1 mg to about 40 mg, about 1 mg toabout 50 mg, about 1 mg to about 60 mg, about 1 mg to about 70 mg, about1 mg to about 80 mg, or about 1 mg to about 90 mg, inclusive, includingany range in between these values, of the active ingredient. The term“unit dosage forms” refers to physically discrete units suitable asunitary dosages for individuals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient or carrier.

The anti-cancer agents (such as oligonucleotide-lased agents) disclosedherein are effective over a wide dosage range and are generallyadministered in a therapeutically effective amount. It will beunderstood, however, that the amount of the anti-cancer agents actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient anticancer therapy is mixed with a pharmaceutical excipientor carrier to form a solid preformulation composition containing ahomogeneous mixture of a compound of the present invention. Whenreferring to these preformulation compositions as homogeneous, it ismeant that the active ingredient is dispersed evenly throughout thecomposition so that the composition can be readily subdivided intoequally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage than affording the advantage of prolongedaction and to protect the anticancer therapies (such as anoligonucleotide) from acid hydrolysis in the stomach. For example, thetablet or pill can comprise an inner dosage and an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permit the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

The liquid forms in which the compositions of the present invention canbe incorporated for administration orally or by injection includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as corn oil, cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Parenteral routes of administration include but are not limited todirect injection into a central venous line, intravenous, intramuscular,intraperitoneal, intradermal, or subcutaneous injection. Oligonucleotide(e.g., an oliconucleotide and microcarrier formulation) formulationssuitable for parenteral administration are generally formulated in USPwater or water for injection and may further comprise pH buffers, saltsbulking agents, preservatives, and other pharmaceutically acceptableexcipients. Oligonucleotide(s), for example as oligonucleotidemicrocarrier complexes or encapsulates, for parenteral injection may heformulated in pharmaceutically acceptable sterile isotonic solutionssuch as saline and phosphate buffered saline for injection.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions cancontain suitable pharmaceutically acceptable excipients as describedherein. The compositions can be administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpharmaceutically acceptable solvents can be nebulized by use of inertgases. Nebulized solutions can be inhaled directly from the nebulizingdevice or the, nebulizing device can be attached to a face mask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions can also be administered, orally or nasally, fromdevices which deliver the formulation in an appropriate manner.

V. Methods of Treatment A. Methods for Suppressing or PreventingMetastasis of a Cancer

In one aspect, provided herein is one or more oligonucleotide (orcomposition thereof) for use in suppressing or preventing metastasis ofa cancer in an individual. In another aspect, provided herein is one ormore oligonucleotide (or compositions thereof) for use in combinationwith at least one therapy for suppressing or preventing metastasis of acancer in an individual. In any of the aspects, herein, the individualmay have been previously treated for cancer with a therapy.

In some aspects, the invention provides a method for suppressingmetastasis of a cancer in an individual comprising administering to theindividual an effective amount of one or more oligonucleotide describedherein, wherein the oligonucleotide is able to hybridize with thechimeric mitochondrial RNA molecules to form a stable duplex, andwherein the individual has been previously treated for cancer with atherapy. In a further embodiment, the method for suppressing metastasisof a cancer in an individual comprises administering the one or moreoligonucleotide in combination with at least one therapy disclosedherein. In some embodiments, the at least one therapy is selected fromthe group consisting of an anti-cancer agent, a radiation therapy,surgery, an allogenic stem cell transplant therapy, and an autologousstem cell transplant therapy. In some of the embodiments herein, theindividual has been previously treated for cancer with a therapycomprising chemotherapy, radiation therapy, surgery, or combinationsthereof. In some embodiments, the individual has been previously treatedwith one or more of bortezomib, cyclophosphamide, dexamethasone,doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylatedinterferon-alpha, prednisone, thalidomide, and vincristine. In any ofthe embodiments herein, the oligonucleotide and the at least one therapyis administered sequentially. For example, one or more oligonucleotidedescribed herein can be administered to an individual before or after atumor(s) has been surgically resected from the individual. In someembodiments, the oligonucleotide and the at least one therapy isadministered simultaneously. For example, one or more oligonucleotidedescribed herein can be administered to an individual during surgicalresection of a tumor(s) from the individual.

In some aspects, the invention provides a method for preventingmetastasis of a cancer in an individual comprising administering to theindividual an effective amount of one or more oligonucleotide describedherein, wherein the oligonucleotide is able to hybridize with thechimeric mitochondrial RNA molecules to form a stable duplex, andwherein the individual has been previously treated for cancer with atherapy. In a further embodiment, the method for suppressing orpreventing metastasis of a cancer in an individual comprisesadministering the one or more oligonucleotide in combination with atleast one therapy disclosed herein. In some embodiments, the at leastone therapy is selected from the group consisting of an anti-canceragent, a radiation therapy, surgery, an allogenic stem cell transplanttherapy, and an autologous stem cell transplant therapy. In some of theembodiments herein, the individual has been previously treated forcancer with a therapy comprising chemotherapy, radiation therapy,surgery, or combinations thereof. In some embodiments, the individualhas been previously treated with one or more of bortezomib,cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha,lenalidomide, melphalan, pegylated interferon-alpha, prednisone,thalidomide, and vincristine. In any of the embodiments herein, theoligonucleotide and the at least one therapy is administeredsequentially. For example, one or more oligonucleotide described hereincan be administered to an individual before or after a tumor(s) has beensurgically resected from the individual. In some embodiments, theoligonucleotide and the at least one therapy is administeredsimultaneously. For example, one or more oligonucleotide describedherein can be administered to an individual during surgical resection ofa tumor(s) from the individual.

As non-limiting examples, a method for suppressing or preventingmetastasis of cancer according to the present invention may be byadministration of one or more oligonucleotide (or a composition thereof)described herein provided as a daily dosage in an amount of about 0.1 toabout 100 mg/kg, such as about 0.5, about 0.9, about 1.0, about 1.1,about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29,about 30, about. 40, about 45, about 50, about 60, about 70, about 80,about 90 or about 100 mg/kg, per day, on at least one of day 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40,or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment,or any combination thereof, using single or divided doses at every 24,12, 8, 6, 4, or 2 hours, or any combination thereof.

In some embodiments, the one or more oligonucleotide (or a compositionthereof) may be administered in combination with at least one therapy(e.g., an anti-cancer agent, a radiation therapy, surgery, an allogenicstem cell transplant therapy, or an autologous stem cell transplanttherapy). In some embodiments, the combination is administeredsequentially. For example, a one or more oligonucleotide describedherein may be administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively, about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20weeks apart from the administration of the at least one therapy duringcombination treatment. In some embodiments, the combination isadministered simultaneously. For example, a one or more oligonucleotidedescribed herein may be administered about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 minutes,or alternatively, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart from theadministration of the at least one therapy during combination treatment.

B. Methods for Treating or Preventing Relapse of a Cancer

In other aspects, provided herein is one or more oligonucleotide (orcompositions thereof) for use in treating or preventing relapse of acancer in an individual. In some embodiments, the individual hasresponded to initial treatment and is in remission.

In some aspects, the invention provides a method for treating orpreventing relapse of cancer in an individual comprising administeringto the individual an effective amount of one or more oligonucleotidedescribed herein, wherein the oligonucleotide is able to hybridize withthe chimeric mitochondrial RNA molecules to form a stable duplex, andwherein the individual has been previously treated for cancer with atherapy. In a further embodiment, the method for treating or preventingrelapse of cancer in an individual comprises administering the one ormore oligonucleotide in combination with at least one therapy disclosedherein. In some embodiments, the at least one therapy is selected fromthe group consisting of an anti-cancer agent, a radiation therapy,surgery, an allogenic, stem cell transplant therapy, and an autologousstem cell transplant therapy. In some of the embodiments herein, theindividual has been previously treated for cancer with a therapycomprising chemotherapy, radiation therapy, surgery, or combinationsthereof. In some embodiments, the individual has been previously treatedwith one or more of bortezomib, cyclophosphamide, dexamethasone,doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylatedinterferon-alpha, prednisone, thalidomide, and vincristine. In any ofthe embodiments herein, the oligonucleotide and the at least one therapyis administered sequentially. For example, one or more oligonucleotidedescribed herein can be administered to an individual before or after atumor(s) has been surgically resected from the individual. In someembodiments, the oligonucleotide and the at least one therapy isadministered simultaneously. For example, one or more oligonucleotidedescribed herein can be administered to an individual during surgicalresection of a tumor(s) from the individual.

As non-limiting examples, a method for treating or preventing relapse ofa cancer according to the present invention may be by administration ofone or more oligonucleotide (or a composition thereof) described hereinprovided as a daily dosage in an amount of about 0.1 to about 100 mg/kg,such as about 0.5, about 0.9, about 1.0, about 1.1, about ILS, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 20, about 21, about 22, about 23, about 24,about 25, about 26, about 27, about 28, about 29, about 30, about 40,about 45, about 50, about 60, about 70, about 80, about 90 or about 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, atleast one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 after initiation of treatment, or any combinationthereof, using single or divided doses at every 24, 12, 8, 6, 4, or 2hours, or any combination thereof.

In some embodiments, the one or more oligonucleotide (or a compositionthereof) may be administered in combination with at least one therapy(e.g., an anti-cancer agent, a radiation therapy, surgery, an allogenicstem cell transplant therapy, or an autologous stem cell transplanttherapy). In some embodiments, the combination is administeredsequentially. For example, a one or more oligonucleotide describedherein may be administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively, about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20weeks apart from the asrninistration of the at least one therapy duringcombination treatment. In some embodiments, the combination isadministered simultaneously. For example, a one or more oligonucleotidedescribed herein may be administered about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 minutes,or alternatively, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart from theadministration of the at least one therapy during combination treatment.

In other embodiments, a “maintenance schedule” may be used in which oneor more maintenance oligonucleotide-based (such as antisense-based)therapies are administered less frequency than in the original treatmentadministered prior to remission, such as once per week or once every twoweeks. The maintenance schedule can be continued either for a fixedperiod of time, generally about 1 or about 2 years, or indefinitely aslong as the patient is continuing to show no signs of progressivedisease and is tolerating the treatment without significant toxicity.

C. Methods for Treating Metastatic Cancer

In yet other aspects, provided herein is one or more oligonucleotide (orcompositions thereof) for use in treating metastatic cancer (such asrelapsed metastatic cancer) in an individual.

In some aspects, the invention provides a method for the treatment ofmetastatic cancer (such as relapsed metastatic cancer) in an individualcomprising administering to the individual an effective amount of one ormore oligonucleotide described herein, wherein the oligonucleotide isable to hybridize with the chimeric mitochondrial RNA molecules to forma stable duplex, and wherein the individual has been previously treatedfor cancer with a therapy. In a further embodiment, the method fortreating metastatic cancer (such as relapsed metastatic cancer) in anindividual comprises administering the one or more oligonucleotide incombination with at least one therapy disclosed herein. In someembodiments, the at least one therapy is selected from the groupconsisting of an anti-cancer agent, a radiation therapy, surgery, anallogenic stem cell transplant therapy, and an autologous stem celltransplant therapy. In some of the embodiments herein, the individualhas been previously treated for cancer with a therapy comprisingchemotherapy, radiation therapy, surgery, or combinations thereof In anyof the embodiments herein, the oligonucleotide and the at least onetherapy is administered sequentially. For example, one or moreoligonucleotide described herein can be administered to an individualbefore or after a tumors) has been surgically resected from theindividual. In some embodiments, the oligonucleotide and the at leastone therapy is administered simultaneously, For example, one or moreoligonucleotide described herein can be administered to an individualduring surgical resection of a tumor(s) from the individual.

As non-limiting examples, a method for the treatment of metastaticcancer (such as relapsed metastatic cancer) according to the presentinvention may be by administration of one or more oligonucleotide (or acomposition thereof) described herein provided as a daily dosage in anamount of about 0.1 to about 100 mg/kg, such as about 0.5, about 0.9,about 1.0, about 1.1, about 1.5, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, about 30, about 40, about 45, about 50, about60, about 70, about 80, about 90 or about 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses at every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments, the one or more oligonucleotide (or a compositionthereof) may be administered in combination with at least one therapy(e.g., an anti-cancer agent, a radiation therapy, surgery, an allogenicstem cell transplant therapy, or an autologous stein cell transplanttherapy). In some embodiments, the combination is administeredsequentially. For example, a one or more oligonucleotide describedherein may be administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively, about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19 or 20weeks apart from the asministration of the at least one therapy duringcombination treatment. In some embodiments, the combination isadministered simultaneously. For example, a one or more oligonucleotidedescribed herein may be administered about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 minutes,or alternatively, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart from theadministration of the at least one therapy during combination treatment.

In another embodiment, the therapeutically effective amount of said oneor more oligonucleotide (or compositions thereof) is administered aspart of a salvage therapy in treating an individual wherein the cancerhas become refractory to other treatment for cancer. In someembodiments, the individual relapsed after treatment with one or more ofbortezomib, cyclophosphamide, dexamethasone, doxorubicin,interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha,prednisone, thalidomide, and vincristine.

Without being bound by theory, it is believed that CSCs give rise to therelapse and/or metastasis of a cancer following treatment of the primarytumor. In some aspects, methods of treatment as described herein (e.g.,a method for suppressing or preventing metastasis of a cancer, etc.)eliminates or suppresses cancer stem cells. In some embodiments, theoligonucleotides disclosed herein can kill or inhibit cancer stem cells(CSCs) to suppress metastasis, prevent metastasis or prevent relapse ofa cancer in an individual. In some embodiments, the individual has beenpreviously treated for cancer with a therapy. In one embodiment, theoligonucleotides disclosed herein can kill or inhibit cancer stem cells(CSCs) that are resistant to treatment (e.g., chemotherapy). In oneembodiment, treatment of an individual with any one or moreoligonucleotide disclosed herein (e.g., an oligonucleotide complementaryto an ASncmtRNA molecule) non-selectively inhibits, arrests, kills, orabolishes the CSCs in the individual. In one embodiment, any one or moreoligonucleotide disclosed herein (e.g., an oligonucleotide complementaryto an ASncmtRNA molecule) reduces the number of CSCs in the individualas compared to an individual not administered the oligonucleotide. In afurther embodiment, the individual has been previously treated forcancer with a therapy.

In any embodiments of the methods herein, any one or moreoligonucleotide disclosed herein (e.g., an oligonucleotide complementaryto an ASncmtRNA molecule) inhibits tumor growth and/or metastasis in theindividual as compared to an individual not administered theoligonucleotide.

In any of the embodiments of the methods herein (e.g., a method forsuppressing or preventing metastasis of a cancer, a method for treatingor preventing relapse of a cancer, a method for treating metastaticcancer, etc.), the cancer may be a solid cancer or a non-solid cancer.In any of the embodiments herein, the cancer is a solid cancer. Examplesof solid cancers contemplated herein include, without limitation,squamous cell cancer, small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, brain cancer, cervical cancer,ovarian cancer, liver cancer, sarcoma, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, oralpharyngeal cancer, salivary gland carcinoma, renalcancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, gastric cancer, melanoma, and various types of headand neck cancer.

In some embodiments, the cancer is associated with a human papillomavirus (HPV) infection, also referred to herein as “HPV-associatedcancer”, such as in cervical cancer, oralpharyngeal cancer, and head andneck cancer. For example, one of the most important risk factors fordevelopment of cervical cancer is an HPV infection. Over 100 strains ofHPV have been identified, however, only a subset are classified ashigh-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and82) or probable high-risk (26, 53, and 66) types for the development ofcancer (Munoz et al., NEJM, 348:518-527, 2003). Of these HPV types,HPV16 and HPV18 are reported to cause nearly 70% of all cervical cancercases while HPV 31 and 35 cause another 10% of cervical cancer cases.See Walboomers et al., J Pathol., 189(1):12-9, 1999. HPV-associatedcancer can involve one or more of the following steps: (1) initial HPVinfection, (2) persistent FIPV infection, (3) transforming HPVinfection, in the presence or absence of integration of HPV DNA into thehost cell genome, (4) development of precancerous lesions, (5)development of at least one primary tumor, and (6) development ofinvasive cancer (e.g., metastatic cancer).

In some instances, the HPV-associated cancer is resistant tochemotherapeutic agents regularly used for the treatment of cancer. Forexample, HPV 16-immortalized cervical cells can develop resistance tocisplatin, paclitaxel, actinomycin D, doxrubucin, etoposide, and5-fluorouracil which presents a major obstacle in cancer treatment. SeeDing et al., Int J Cancer, 15:87(6)818-23, 2000. in some embodimentsherein, provided herein is one or more oligonucleotide (or compositionsthereof) for use in suppressing metastasis of a cancer in an individual,wherein the cancer is resistant to a chemotherapeutic agent, and whereinthe cancer is an HPV-associated cancer. In some embodiments herein,provided herein is one or more oligonucleotide (or compositions thereof)for use in treating or preventing relapse of a cancer in an individual,wherein the cancer is resistant to a chemotherapeutic agent, and whereinthe cancer is an HPV-associated cancer. some embodiments herein,provided herein is one or more oligonucleotide (or compositions thereof)for use in treating metastatic cancer (such as relapsed metastaticcancer) in an individual, wherein the metastatic cancer is resistant toa chemotherapeutic agent, and wherein the metastatic cancer is anHPV-associated cancer. In some embodiments herein, provided herein isone or more oligonucleotide (or compositions thereof) for use intreating a refractory cancer in an individual. In some embodiments, therefractory cancer is a refractory HPV-associated cancer. In someembodiments, the refractory HPV-associated cancer is resistant to achemotherapeutic agent. As used herein, the term “refractory cancer”refers to a cancer (e.g., an HPV-associated cancer) that does notrespond to treatment, for example, a cancer that is resistant at thebeginning of treatment (e.g., treatment with a chemotherapeutic agent)or a cancer that may become resistant during treatment. In someembodiments, the chemotherapeutic agent is selected from the groupconsisting of cisplatin, paclitaxel, actinomycin D, doxrubucin,etoposide, and 5-fluorouracil. In some embodiments, the chemotherapeuticagent is cisplatin. In some of the embodiments herein, theHPV-associated cancer (e.g., a refractory HPV-associated cancer) is froman infection with one or more HPV strains selected from the groupconsisting of HPV 16, HPV 18, HPV 31 and HPV 45. In some of theembodiments herein, the HPV-associated cancer (e.g., a refractoryHPV-associated cancer) is from an infection with the HPV strain HPV 45.In some embodiments, the one or more oligonucleotide is complementary tothe SncmtRNA molecule encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In someembodiments, the one or more oligonucleotide is complementary to theASncmtRNA molecule encoded by a nucleotide sequence selected from thegroup consisting, of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In someembodiments, the one or more oligonucleotide comprises a nucleotidesequence selected from the group consisting of SEQ NOs:7-198. In someembodiments, the one or more oligonucleotide comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:36, 197 and198.

In some embodiments, provided herein is one or more oligonucleotide (orcompositions thereof) for use in treating a refractory cancer in anindividual. In some embodiments, the refractory cancer is resistant to achemotherapeutic agent. In some embodiments, the chemotherapeutic agentis cisplatin. In some embodiments, the refractory cancer is a solidcancer disclosed herein. For example, the refractory solid cancer may beone or more of bladder cancer, brain cancer, breast cancer, cervicalcancer (e.g., a refractory HPV-associated cervical cancer), coloncancer, endometrial cancer, esophageal cancer, gastric cancer, liver andbile duct cancer, lung cancer, melanoma, oral cancer, ovarian cancer,pancreatic cancer, pharynx cancer, prostate cancer, renal cancer,testicular cancer, or thyroid cancer. In some embodiments, therefractory cancer is a non-solid cancer disclosed herein. For example,the refractory cancer may be one or more of multiple myeloma, leukemia,or lymphoma.

In any of the embodiments herein, the cancer is a non-solid cancer.“Non-solid cancer” refers to a hematological malignancy involvingabnormal growth and/or metastasis of a blood cell. Examples of non-solidcancers contemplated herein include, without limitation, multiplemyeloma, acute myeloid leukemia, acute lymphoblastic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, acute nonlymphocyticleukemia, acute granulocytic leukemia, chronic granulocytic leukemia,acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,bovine leukemia, chromic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukenna,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, undifferentiated cell leukemia, idiopathic myelofibrosis,lymphoma (such as Non-Hodgkin's lymphoma, and Hodgkin's lymphoma), andmyelodysplastic syndrome.

The methods disclosed herein can be practiced in an adjuvant setting.“Adjuvant setting” can refers to a clinical setting in which anindividual has had a history of cancer, and generally (but notnecessarily) been responsive to therapy, which includes, but is notlimited to, surgery (such as surgical resection), radiotherapy, andchemotherapy. However, because of their history of the cancer (such asmelanoma or colon cancer), these individuals are considered at risk ofdevelopment of cancer. Treatment or administration in the “adjuvantsetting” refers to a subsequent mode of treatment. The degree of risk(i.e., when an individual in the adjuvant setting is considered as “highrisk” or “low risk”) depends upon several factors, most usually theextent of disease (cancer) when first treated.

The present invention is accordingly directed to methods for inhibitingthe symptoms or conditions (disabilities, impairments) associated withcancer (e.g., metastatic cancer or relapsed cancer) as described indetail below. As such, it is not required that all effects of thecondition be entirely prevented or reversed, although the effects of thepresently disclosed methods likely extend to a significant therapeuticbenefit for the individual. As such, a therapeutic benefit is notnecessarily a complete prevention or cure for the condition, but rather,can encompass a result which includes reducing or preventing thesymptoms that result from cancer (e.g., metastatic cancer or relapsedcancer), reducing or preventing the occurrence of such symptoms (eitherquantitatively or qualitatively), reducing the severity of such symptomsor physiological effects thereof, and/or enhancing the recovery of theindividual after experiencing cancer (e.g., metastatic cancer orrelapsed cancer) symptoms.

Specifically, the therapies (e.g., one or more oligonucleotide) of thepresent invention, when administered to an individual, can treat orprevent one Or more of the symptoms or conditions associated with cancer(e.g., metastatic cancer or relapsed. cancer) and/or reduce or alleviatesymptoms of or conditions associated with this disorder. As such,protecting an individual from the effects or symptoms resulting fromcancer (e.g., metastatic cancer or relapsed cancer) includes bothpreventing or reducing the occurrence and/or severity of the effects ofthe disorder and treating a patient in which the effects of the disorderare already occurring or beginning to occur. A beneficial effect caneasily be assessed by one of ordinary skill in the art and/or by atrained clinician who is treating the patient. Preferably, there is apositive or beneficial difference in the severity or occurrence of atleast one clinical or biological score, value, or measure used toevaluate such individual in those who have been treated with the methodsof the present invention as compared to those that have not. Forexample, the at least one clinical or biological score, value, ormeasure used to evaluate such an individual is the capability of cells(e.g., cancer stem cells) taken from a primary tumor, a secondary tumor,a biopsy, or ascites fluid of an individual to form spheres in a sphereformation assay. Methods for purifying cells such as cancer stem cellsfrom tumors or other biological samples are well known in the art suchas in U.S. Pat. No. 8,614,095, the disclosure of which is incorporatedby reference herein in its entirety. In an exemplary sphere formationassay, a tumor is surgically removed from a subject and minced with ascalpel into fragments of approximately 2 to 3 mm3. The fragments arewashed with a buffer (e.g., PBS) and then incubated with buffercontaining sodium hypochlorite. The tumor tissue fragments are washedwith buffer and digested with PBS and digested with a medium containingone or more of collagenase I, collagenase IV, dispase, hyaluronidase andDNAase. The cell suspension is centrifuged and the pellet is suspendedin buffer containing βFGF and EGF. The cells are washed to remove serumand suspended in medium supplemented with human EGF, human βFGF, B27supplement without vitamin A, hydrocortisone, insulin, and N2supplement. The cells are subsequently cultured in non-adherent plates.After 10 days in culture, spheres of 100 to 200 μm in diameter areobtained and counted. The spheres can be further expanded clonally, orinjected into a subject to observe the capability of tumor formation. Insome embodiments, an individual that has received an effective amount ofone or more oligonucleotide (or compositions thereof) disclosed herein,alone or in combination with at least one therapy disclosed herein, hasreduced sphere formation as compared to an individual not treated withthe oligonucleotides of the present invention.

VI. Articles of Manufacture or Kits

In another aspect, an article of manufacture or kit is provided whichcomprises one or more oligonucleotide described herein. The article ofmanufacture or kit may further comprise instructions for use of the oneor more oligonucleotide in the methods of the invention. Accordingly, incertain embodiments, the article of manufacture or kit comprisesinstructions for use of one or more oligonucleotide complementary to anantisense non-coding chimeric mitochondrial RNA (ASncmtRNA) molecule ora sense non-coding chimeric mitochondrial RNA (SncmtRNA) molecule inmethods for suppressing metastasis of a cancer, preventing or treatingrelapse of a cancer, and/or treating metastatic cancer in an individualcomprising administering to the individual an effective amount of theone or more oligonucleotide. In certain embodiments, the individual hasbeen previously treated for cancer with a therapy (e.g., chemotherapy,radiation therapy, surgery, or combinations thereof). In someembodiments, the article of manufacture or kit comprises instructionsfor use of one or more oligonucleotide complementary to an antisensenon-coding chimeric mitochondrial RNA (ASncmtRNA) molecule or a sensenon-coding chimeric mitochondrial RNA (SncmtRNA) molecule in methods fortreating a refractory cancer (e.g., a refractory HPV-associated cancer)in an individual comprising administering to the individual an effectiveamount of the one or more oligonucleotide.

The article of manufacture or kit may further comprise a container.Suitable containers include, for example, bottles, vials (e.g., dualchamber vials), syringes (e.g., single or dual chamber syringes), IVbags, and test tubes. The container may be formed from a variety ofmaterials such as glass or plastic. The container holds the composition(e.g., pharmaceutical formulation).

The article of manufacture or kit may further comprise a label orpackage insert, which is on, or associated with the container and mayindicate directions for reconstitution and/or use of the composition(e.g., pharmaceutical formulation). The label may further indicate thatthe formulation is useful or intended for intravenous, subcutaneous, orother modes of administration for suppressing metastasis of a cancer,preventing or treating relapse of a cancer, and/or treating metastaticcancer in an individual. In other embodiments, the label may furtherindicate that the formulation is useful or intended for intravenous,subcutaneous, or other modes of administration for treating a refactorycancer (e.g., a refractory HPV-associated cancer) in an individual. Thecontainer holding the formulation may he a single-use vial or amulti-use vial, which allows for repeat administrations (e.g., from 2-6administrations) of the reconstituted composition (e.g., pharmaceuticalformulation). The article of manufacture or kit may further comprise asecond container comprising a suitable diluent. The article ofmanufacture or kit may further include other materials desirable from acommercial, therapeutic, and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

The article of manufacture of kit described herein optionally furthercomprises a container comprising a second therapeutic composition (e.g.,an anti-cancer agent). For example, the article of manufacture or kitcan comprise one or more oligonucleotide as a first composition (e.g., afirst pharmaceutical composition) and an anti-cancer agent as a secondcomposition (e.g., a second pharmaceutical composition). In someembodiments, the kit further comprises instructions for use of the oneor more oligonucleotide in combination with the anti-cancer agent in themethods of the invention. An exemplary anti-cancer agents may beremicade, docetaxel, celecoxib, melphalan, dexamethasone, steroids,gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide,temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan,methotrexate, gefitinib, taxol, taxotere, fluorouracil, leucovorin,irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferonalpha, capecitabine, cisplatin, thiotepa, fludarabine, carboplatin,liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine,IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate,biaxin, busulphan, prednisone, bortezomib, bisphosphonate, arsenictrioxide, vincristine, doxorubicin, paclitaxel, ganciclovir, adriamycin,estrainustine sodium phosphate, sulindac, and/or etoposide.

VII. Additional Exemplary Embodiments

The present application in some embodiments provides a method oftreatment for a refractory cancer in an individual comprisingadministering to the individual an effective amount of one or moreoligonucleotide complementary to an antisense non-coding chimericmitochondrial RNA (ASncmtRNA) molecule or a sense non-coding chimericmitochondrial RNA (SncmtRNA) molecule, wherein the oligonucleotide isable to hybridize with the chimeric mitochondrial RNA molecules to forma stable duplex.

In sonic embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide is sufficiently complementary toa human non-coding chimeric mitochondrial RNA molecule comprising: a) anantisense 16S mitochondrial ribosomal RNA covalently linked at its 5′end to the 3′ end of a polynucleotide with an inverted repeat sequenceor b) a sense 16S mitochondrial. ribosomal RNA covalently linked at its5′ end to the 3′ end of a polynucleotide with an inverted repeatsequence.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide is complementary to the ASncmtRNAmolecule encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide is at least 85% complementary tothe ASncmtRNA molecule encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

In some embodiments according to (or as applied to) any of theembodiments above, the one or more oligonucleotide comprises anucleotide sequence selected from the group consisting of SEQ IDNOs:7-198.

In some embodiments according to (or as applied to) any of theembodiments above, the one or more oligonucleotide comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs:36, 197and 198.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide is administered in combinationwith at least one anti-cancer agent.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide and the at least one anti-canceragent is administered sequentially.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide and the at least one anti-canceragent is administered simultaneously.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide is administered in combinationwith a radiation therapy.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide is administered in combinationwith surgery.

In some embodiments according to (or as applied to) any of theembodiments above, the individual has been previously treated for cancerwith a therapy comprising chemotherapy, radiation therapy, surgery, orcombinations thereof.

In some embodiments according to (or as applied to) any of theembodiments above, the refractory cancer is a refractory HPV-associatedcancer.

In some embodiments according to (or as applied to) any of theembodiments above, the refractory HPV-associated cancer is one or moreselected from the group consisting of: cervical cancer, oralpharyngealcancer, and head and neck cancer.

In some embodiments according to (or as applied to) any of theembodiments above, the refractory HPV-associated cancer is resistant toa chemotherapeutic agent.

In some embodiments according to (or as applied to) any of theembodiments above, the chemotherapeutic agent is one or more selectedfrom the group consisting of: cisplatin, paclitaxel, actinomycin D,doxrubucin, etoposide, and 5-fluorouracil.

In some embodiments according to (or as applied to) any of theembodiments above, the refractory HPV-associated cancer is from aninfection with one or more HPV strains selected from the groupconsisting of: HPV 16, HPV 18, HPV 31 and HPV 45.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide reduces the number of cancer stemcells in the individual as compared to an individual not administeredthe oligonucleotide.

In some embodiments according to (or as applied to) any of theembodiments above, the oligonucleotide inhibits tumor growth and/ormetastasis in the individual as compared to an individual notadministered the oligonucleotide.

The invention will be more fully understood by reference to thefollowing examples. The examples, which are intended to be purelyexemplary of the invention, should not be construed as limiting thescope of the invention in any way. It is understood that the examplesand embodiments described herein are for illustrative purposes only andthat various modifications or changes in light thereof will be suggestedto persons skilled in the art and are to be included within the spiritand purview of this application and scope of the appended claims.

EXAMPLES Example 1: Treatment of HCT-116 Colon Cancer Cells and PrimaryCultures of Human Colon Cancer Cells with Antisense OligonucleotidesComplementary to Antisense Non-Coding Chimeric Mitochondrial RNAAbolished Sphere Formation

In this study, the ability of cells, from both the HCT-116 colon cancercell line and primary cultures of human colon cancer cells derived frompatients, to form spheroid bodies following treatment with antisenseoligonucleotides directed to antisense non-coding chimeric mitochondrialRNA (ASncmtRNA) was determined. The assay utilized measured the numbersof spheroid bodies, also referred to herein as spheres, based on thespecific ability of cancer stem cells to form these spheroid bodies.

Materials and Methods Experimental Scheme

FIG. 1 demonstrates a general scheme of the experimental procedure andthe assay utilized herein to measure the effect of antisenseoligonucleotides targeted to ASncmtRNA on the number of spheres formedin colon cancer cells, based on the specific ability of cancer stemcells to form these spheroid bodies. Sphere formation was measured inHCT-116 colon cancer cells and primary cultures of human colon cancercells derived from patients.

Primary Cultures of Human Cancer Cells

Biopsies of colon cancer were received post-surgery and withcorresponding informed consent of each patient. Pieces of tumors ofabout 500 mm3 were transferred to sterile tubes of 50 ml containing DIEMmedium. High-Glucose, GlataMax™ (GIBCO), 10% FCS (BiologicalIndustries), Fungizone® 1×, 2× antibiotic-antimycotic mix and gentamicin25 μg/ml (Invitrogen). The biopsies were processed within 2 to 3 hpost-surgery.

In order to disaggregate the tumor, a piece of colon tumor was mincedwith a scalpel into fragments of approximately 2 to 3 mm3. The fragmentswere washed twice with PBS and then incubated for 20 minutes with PBScontaining 0.5% sodium hypochlorite. The fragments were washed threetimes with PBS and digested with RPMI medium (Invitrogen) containing 1mg/ml collagenase 1, 2mg/ml collagenase IV, 1 mg/ml dispase, 20 μg/mlhyaluronidase and 2000 U/ml DNAase. The mix was incubated at 37° C. for60 min and with constant stirring. Next, the cell suspension wascentrifuged at 200×g for 5 minutes and the pellet was suspended in PBSand centrifuged again a 200×g for 5 minutes. The pellet was resuspendedin DMEM/F12 containing 1×N2 supplement, 10 ng/ml βFGF and 20 ng/ml EGF,for the formation of spheres.

Culture of HCT-116 Cells

HCT-116 cells were cultured according to standard protocols. HCT-116cells obtained from ATCC were grown in DMEM medium containingpenicillin, gentamicin and fungizone, with 10% FCS, at 37° C. and with5% CO2.

Selection of Tumor Cells by Sphere Formation and Adherence to CoatedPlates

Cells derived from colon cancer tumors or HCT-116 cultures werecollected and washed to remove serum and suspended in serum-freeDMEM/E12 supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin,2.0 ng/ml human EGF, 20 ng/ml human βFGF, 2% B27 supplement withoutvitamin A, hydrocortisone, insulin (Lonza) and N2 supplement(Invitrogen, Carlsbad, Calif., USA). The cells were subsequentlycultured in non-adherent plates (Corning Inc., Corning, N.Y., USA) at adensity of about 5,000 cells/well or 1×105 cells in T25 flasks (CorningInc. T25 3815). After 10 days in culture, spheres of 100 to 200 μm indiameter were obtained. The medium containing the spheres were filteredusing a 70 μm nylon filter to eliminate single cells. The spheres werecollected and dissociated with trypsin-EDTA and mechanically disruptedwith a pipette. The cells were then centrifuged to remove the enzyme,washed once with DMEM medium containing 105 CES and plated in adherentplates coated with collagen I (Gibco) and cultured at 37° C. and 5% CO2as described before.

Cell Transfection and Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) used in this study were synthesized byIDT (integrated DNA Technologies, USA), Invitrogen or Biosearch Inc.with 100% phosphorothioate (PS) internucleosidic linkages. Fortransfection, cells were seeded into 12-well plates (Nunc) at 50,000cells/well. The next day, cells selected from colon cancer tumors orfrom HCT-116 cultures (see Selection of tumor cells by sphere formationand adherence to coated plates) were transfected with the antisenseoligonucleotides (ASO 11017S: 5′-GTCCTAAACTACCAAACC-3′ (SEQ ID NO:197)or ASO 1537S: 5′-CACCCACCCAAGAACAGG-3′ (SEQ ID NO:36), depending on thecell type) or a control oligonucleotide (Control Oligo 154:5′-AGGTGGAGTGGATTGGGG-3′ (SEQ ID NO:199)) at a final concentration of100 to 200 nM (depending on the cell type) using Lipofectamine 2000(Invitrogen) according to the manufacturer's directions. In addition, asubset of cells were left untreated or in the presence of onlyLipofectamine 2000. Transfection was for 48 hours under normal cultureconditions.

Sphere Formation Assay

At 48 hours post transfection, the cells were harvested, counted and5,000 to 6,000 cells were cultured in non-adherent 6-well plates(Corning Inc., Corning, N.Y., USA) as described before (describedsupra). After 10 days in culture, spheres of 100 to 200 μm in diameterwere obtained and counted.

Results

Sphere formation was unchanged in primary cultures of colon tumor cellsfollowing no treatment, treatment with only Lipofectamine, or treatmentwith Control Oligo 154. But sphere formation was abolished in theseprimary cells following treatment with ASO 1537S (FIG. 2).

A primary culture of a colon tumor (patient TPT; 50,000 cells) was usedto quantify sphere formation and evaluate the effect of transfectionwith ASO 1537S on the capacity of these cells to form spheres. The threegroups of control cells (untreated, treated with Lipofectamine only, ortreated with Control Oligo 154) formed spheres (30 to 37 spheres)equivalent to approximately 0.6% of the total number of cells seeded.Cells transfected with ASO 1537S were unable to form spheres (FIG. 3).

Sphere formation was unchanged in cells from the HCT-116 colon tumorcell line following no treatment, treatment with only Lipofectamine, ortreatment with Control Oligo 154. However, sphere formation wasabolished in these cells following treatment with ASO 1107S (FIG. 4).

The colon tumor cell line HCT-116 was used to quantify sphere formationand evaluate the effect of transfection with ASO 1107S on sphereformation. The three groups of control cells (untreated, treated withLipofectamine only, or treated with Control Oligo 154) formed spheres(100 to 160 spheres), equivalent to 0.3% of the total amount of cellsseeded (in this representative example 50,000 cells in T25 flasks).However, cells transfected with ASO 1107S were not able to form spheres(FIG. 5).

Taken together, these results show that groups left untreated, treatedwith only Lipofectamine, or transfected with a control oligonucleotideretained the ability to form spheres. In contrast, primary colon tumorcells or HCT-116 colon cancer cells transfected with antisenseoligonucleotides lost their ability to form spheres. These resultsindicate that the antisense oligonucleotides were able to kill cancerstem cells as indicated by the lack of sphere formation upon treatment.Furthermore, these results also indicate that only a fraction of allcells seeded were able to form spheres.

Example 2: Treatment of a Cervical Cancer Cell Line and Primary Culturesof Human Uterine Cervical Cancer Cells with Antisense OligonucleotidesComplementary to Antisense Non-Coding Chimeric Mitochondrial RNAAbolished Sphere Formation

The ability of cervical cells from the SiHa cervical cancer cell line(transformed with Human Papillomavirus 16 or HPV 16) and from primarycultures of human uterine cervical cancer cells derived from patients toform spheroid bodies following treatment with antisense oligonucleotidesdirected to antisense non-coding chimeric mitochondrial RNA (ASncmtRNA)was determined. The assay utilized measured the numbers of spheroidbodies, also referred to herein as spheres, which ared formed by cancerstem cells.

Materials and Methods Experimental Scheme

The experimental procedure and assay utilized herein measured the effectof antisense oligonucleotides that were targeted to ASncmtRNA on thenumber of spheres formed by cervical cancer cells (FIG. 6). Sphereformation was measured in the SiHa cervical cancer cell line and primarycultures of human cervical. cancer cells derived from patients (CerCa).CerCa 1, CerCa 2 and CerCa 3 (transformed with Human Papillomavirus 45)were obtained from patient biopsies.

Culture of SiHa Cells

SiHa cells were grown in DMEM, “High Glucose”, GlutaMAX™, containingpenicillin, gentamicin and fungizone, with 10% FCS at 37° C. and with 5%CO2.

Primary Cultures of Human Cancer Cells

Biopsies of uterine cervical cancer were received post-surgery and withthe corresponding informed consent of each patient. Pieces of tumors ofabout 500 mm3 were transferred to 50 mL sterile tubes containing DMEMmedium, High-Glucose, GlataMax™ (GIBCO), 10% FCS (Biologicalindustries), Fungizone® 1×, 2× antibiotic-antimycotic mix and gentamicin25 μg/ml (Invitrogen). The biopsies were processed within 2 to 3 hourspost-surgery.

In order to disaggregate the tumor, the tissue sample was placed on a 10cm Petri dish with 10 ml of sterile PBS (Gibco) and sliced with ascalpel into small pieces (1-3 mm3). The pieces were transfered to 6 cmdishes together with 2 ml of medium containing 0.5% Colagenase I, 0.5%Colagenase H, 0.5% Colagenase IV (GIBCO), 0.5% hialorunidase(AppliChem), 0.1% Dispase (GIBCO)), 0.05% DNase (AppliChem), 0.1% BSA(Rockland), antibiotic-antimycotic 2× mix (GIBCO) and Fungizone® 2×(GIBCO). The fragments were incubated at 37° C. for 30 min and the cellsuspension was centrifuged at 300×g for 5 minutes, The pellet wassuspended in 5 ml MEGM™ medium containing 20 ng/ml of hEGF 20,hydrocortisone, insulin, GA-1000 (Lonza), 0.5× B-27 without vitamin A(Invitrogen), 20 ng/ml of FGFb (Invitrogen) and 5% CFB. The cells werecultured in a T25 flask coated with collagen (BD Bioscience) at 37° C.and with 5% CO2. The medium was changed every 48 hours.

Selection of Tumor Cells by Sphere Formation and Adherence to CoatedPlates

About 1×105 cells resuspended in MEGM™ medium supplemented with 20 ng/mlof hEGF, hydrocortisone, insulin, GA-1000 (Lonza), 0.5× B-27 withoutvitamin A (invitrogen), 20 ng/ml of FGFb (Invitrogen) without CFS wereseeded on ultra-low adherence plates (Corning). Ten days later, thespheres were counted under phase microscopy, collected and filteredusing a nylon mesh of 70 μm to discard single cells. The spheres wererecovered from the filter and seeded on 6-well plates (Corning)previously coated with collagen type I (Gibco) and cultured as describedabove. Three human primary cultures of cervical tumors, CerCa 1, CerCa 2and CerCa 3, were isolated.

Human Papillomnavirus (HPV) Genotyping

The different primary cultures were analyzed with the PCMY09/11 LinearArray (Roche). The presence of HPV 16 was detected in CerCa 1 and CerCa2 primary cell cultures while HPV 45 was detected in the CerCa 3 primarycell culture.

Cell Transfection and Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) used in this study were synthesized byIDT (integrated DNA Technologies, USA), invitrogen or Biosearch Inc.with 100% phosphorothioate (PS) internucleosidic linkages. Fortransfection, cells were seeded into 12-well plates (Nunc) at 5000cells/well. The next day, cells selected from cervical cancer tumors orfrom SiHa cells (See Selection of Tumor Cells by Sphere Formation andAdherence to Coated Plates) were transfected with an antisenseoligonucleotide (ASC) 1537S: 5′-CACCCACCCAAGAACAGG-3′ (SEQ ID NO:36),depending of the cell type) or a control oligonucleotide 154 (ASO-C:5′-AGGTGGAGTGGATTGGGG-3′(SEQ ID NO:199)) at a final concentration of 100nM (SiHa cells) or 200 nM (CerCa cells) using Lipofectamine 2000(Invitrogen) according to the manufacturer's directions. In addition, asubset of cells were left untreated (NT), transfected in the presence ofLipofectamine 2000 (LIPO) only, or incubated with 45 uM cisplatin(CISP). Transfection was conducted for 72 hours under normal cultureconditions.

Sphere Formation Assay

At 72 hours post transfection, the cells were harvested, counted and5,000 to 6,000 cells were cultured in non-adherent 6--well plates(Corning Inc., Corning, N.Y., USA) as described above. After 10 days inculture, spheres of 100 to 200 μm in diameter were obtained and counted.

Results

Sphere formation was unchanged in primary cultures of cervical tumorcells following no treatment (NT), treatment with only Lipofectamine(LIPO) or treatment with control oligonucleotide 154 (ASO-C). Incontrast, sphere formation was abolished in Sina, CerCa 1, CerCa 2 andCerCa 3 primary cultures following treatment with ASO 1537S (FIG. 7 andFIG. 8). Sphere formation of SiHa, CerCa 1 and CerCa 2 cells (allinfected with HPV 16) was also abolished when cells were treated withthe drug cisplatin (CISP) (45 μM) (FIG. 7 and FIG. 8) while no effectwas observed in CerCa 3 cells (infected with HPV 45) treated withcisplatin.

Taken together, these results show that cervical cancer primary cultures(CerCa 1, CerCa 2 and CerCa 3 cells) and the cell line SiHa leftuntreated, treated with only Lipofectamine, or transfected with acontrol oligonucleotide retained the ability to form spheres. Incontrast, primary cervical tumor cells or SiHa cells transfected withantisense oligonucleotides targeted to the antisense non-coding chimericmitochondrial RNA (ASncmtRNA) molecules or sense non-coding chimericmitochondrial RNA (SncmtRNA) molecule, lost their ability to formspheres whether HPV 16 or HPV 45 positive. These results indicate thatthe antisense oligonucleotides were able to kill cervical cancer stemcells as indicated by the lack of sphere formation upon treatment.Moreover, treatment with the anti-cancer drug cisplatin abolished sphereformation of SiHa, CerCa 1 and CerCa 2 cells (all HPV 16 positive) butnot CerCa 3 that is infected with HPV 45 (FIG. 7 and FIG. 8). Therefore,these results indicate that the antisense oligonuleotides are able tokill cervical cancer stem (CerCa 3 cells) resistant to cisplatintreatment. See Tjalme et al., Am. J. Clin. Pathol., 137:161, 2012; deSanjosé et al., Eur J Cancer., 49(16): 3450, 2013; Tjalma et al., Int JCancer., 132(4):854, 2013.

Example 3: Treatment of Mice with Antisense OligonucleoitidesComplementary to the Antisense Non-Coding Chimeric Mitochondrial RNAFollowing Surgery to Remove Intradermal Melanoma Tumors PreventedRelapse of Tumor Growth and Metastasis in the Lungs and Liver

One common clinical protocol for melanoma includes surgical resectionfollow by systemic administration of drugs. Similar protocols are usedin the practice of other tumors. In the melanoma model presented in thisrepresentative example, B16F10 melanoma cells (100,000 cells in 200 μlof saline) were injected subcutaneously on the back of C57BL/6 mice.About 11 to 12 days post-cell injection, tumors between 700 to 1,000 mm3developed (a 1,000 mm3 tumor in mice is considered equivalent to a 3,000cc3 tumor in humans). At this time, mice were randomly divided in twogroups (Control Oligo ASO 154 and ASO 1560S (SEQ ID NO:198)) withsimilar tumor volume. Tumors were surgically resected under anesthesiaand the wound washed once with 250 μl containing 100 μg of ASO 1560S orASO 154. After surgical suture, a bolus of 200 μl saline containing 100μg of ASO 154 or ASO 1560S was applied into the cavity left by thetumor. Three days post-surgery, mice received on alternative days, 3intravenous (FIG. 9; 1st, 3rd, 5th arrows on timeline) or 3intraperitoneal (FIG. 9; 2nd, 4th, 6th arrows on timeline) injections of250 μl saline containing 100 μg of either ASO 1560S or ASO 154. Tumorgrowth was measured twice a week with a caliper.

Relapse of tumor growth in mice treated with ASO 154 was observed about3 days post-surgery and tumor volume of about 1,500 mm3 was reached onabout the 25th day post-cell injection (FIG. 9). These mice wereeuthanized under anesthesia, and the tumor and other organs were fixedand saved for further studies. No relapse was observed in mice treatedwith ASO 1560S targeted to the mouse ASncmtRNAs. At 130 days post-cellinjection, mice appeared healthy without the presence of detectabletumors and were euthanized to collect organs. Livers and lungs wereanalyzed for the presence of metastatic nodules.

Control mice (treated with Control Oligo ASO 154) showed the presence ofcancer relapse and metastatic black nodules in the lung and liver. Incontrast, the lungs and livers of the mice treated with ASO 1560S lackedthe presence of metastatic nodules demonstrating that ASO 1560Sprevented or suppressed cancer relapse (FIG. 10).

Example 4: Intravenous Treatment of Mice with AntisenseOligonucleoitides Complementary to the Antisense Non-Coding ChimericMitochondrial RNA Following Surgery to Remove Intradermal Kidney TumorsResulted in Absence of Tumor Relapse and Complete Survival

100,000 RENCA cells (ATCC® CRL2947™ mus musculus kidney renaladenocarcina) were injected subcutaneously at day 0. At day 12, tumorsof an average size of 800 mm3 were removed and the site of the tumor waswashed with 100 μg of Control Oligo ASO 154 (4 animals) or ASO 1560S (5animals) in 200 μl, both formulated in liposomes. The animals weresutured and intravenously injected at the site of the removed tumor with100 μg of ASO 154 (FIG. 11, squares) or ASO 1560S (FIG. 11, triangles),both formulated in liposomes, in 250 μl. No further treatment was given.At day 40, (30 days post-surgery), all the control animals had died withtumors of an average size of 1,200 mm3 and extensive metastasis, whileall animals treated with ASO 1560S had no tumors and were still alive atday 61 (FIG. 11).

Example 5: Intraperitoneal Treatment of Mice with AntisenseOligonucleoitides Complementary to the Antisense Non-Coding ChimericMitochondrial RNA Following Surgery to Remove Intradermal Kidney TumorsResulted in Absence of Tumor Relapse and Complete Survival

100,000 RENCA cells (ATCC® CRL-2947™ mus musculus kidney renaladenocarcina) were injected subcutaneously on day 0 in 8 mice. On day 11tumors had an average size of 800 mm3. Tumors of all animals wereremoved by surgery and divided in 2 groups. The wound of the controlgroup was washed once with Control Oligo ASO 154 before suturing and thewound of the treated group was washed with ASO 1560S before suturing.Post-suture, a bolus of 250 μl was intraperiotoneally injected in theplace of where tumor had grown: the control group was injected with ASO154 and the treated group with ASO 1560S. On days 13, 15, 17 and 19,intraperitoneal injections of 25 μg, ASO 154 (FIG. 12, circles) or 25 μgASO 1560S (FIG. 12, squares) formulated in liposomes were injected in avolume of 250 μl. On days 14, 16 and 18, the mice were injectedintravenously with the same protocol. On day 22, all control animals hadtumors larger than 1,200 mm3 and were sacrificed. On day 60, all animalstreated with ASO 1560S had no tumors and were still alive (FIG. 12).

Example 6: Treatment of Mice with Antisense OligonucleoitidesComplementary to the Antisense Non-Coding Chimeric Mitochondrial RNAFollowing Surgery to Remove Intradermal Melanoma Tumors Resulted inAbsence of Tumor Relapse and Complete Survival

B16F10 melanoma cells (100,000 cells in 200 μl of RPMI medium) wereinjected into mice subcutaneously on day 0. On day 11, surgery wascarried out to remove tumors, The tumor volumes varied fromapproximately 800 to 1200 mm3. The wound was washed once.

Post-suture one bolus of 250 μl of oligo in liposomes was injected inthe tumor site. On days 13, 15, 17, 19 and 21, a dose of 25 μg controlASO 154 naked (FIG. 13, squares), 25 μg of control ASO 154 in liposomes(FIG. 13, circles), or 50 μg of ASO 154 in liposomes (FIG. 13,triangles), each in a volume of 250 μl, was intraperitoneally injectedin the mice. Other groups of mice (6 mice per group) were injected in asimilar manner as described above with 50 ƒg of ASO 1560 naked, 25 μg ofASO 1560S in liposomes or 50 μg of ASO 1560S in liposomes (FIG. 13,diamonds).

Compared to mice treated with a control oligonucleotide, post-surgerytreatment with 25 and 50 μg intraperitoneal injections of ASO 1560Sresulted in the absence of the intradermal melanoma tumor relapse andcomplete survival (FIG. 13).

Example 7: Intraperitoneal or Intravenous Treatment of Mice withAntisense Oligonucleotides Complementary to the Antisense Non-CodingChimeric Mitochondrial RNA Following Surgery to Remove SubcutaneousBladder Carcinoma Tumors Resulted in Absence of Tumor and CompleteSurvival

Twelve mice were injected subcutaneously with a 100, 000 MB49 cells(mouse bladder cancer cells). After 15 days mice had tumors of anaverage diameter of 800 mm3. Tumors were surgically removed (day 0) anda bolus of 200 μl containing 100 μg of ASO 154 (control) or ASO 1560S(active drug) was injected at the site of the surgery. Three dayspost-surgery mice were divided in 2 groups and treated intraperitoneallyor intravenously with 100 μl injections containing ASO 154 (controlgroup) or ASO 1560S (treated group) as indicated in FIG. 14. Only onemouse treated intraperitoneally with ASO 1560S developed a tumor. Allthe mice treated intravenously with ASO 1560S remained without tumorsand experienced full survival. Control animals treated with ControlOligo ASO 154 were sacrificed at day 41 (FIG. 14).

Example 8: Treatment of Rae -/- Mice with Antisense OligonucleotidesComplementary to the Antisense Non-Coding Chimeric Mitochondrial RNAFollowing Tumor Removal of a Human Melanoma Resulted in SignificantElimination of Tumor Relapse and a Large increase in Survival

Rag -/- mice were injected with 5 million human A375 melanoma cells.Approximately at 34 days post cell-injection, all mice developed tumorsof about 700 mm3. Mice were subjected to tumor removal surgery anddivided randomly in two groups.

Group 1 (control): the wound was washed with 50 μg of Control Oligo 154(6 mice in group) in a volume of 200 μl. Group 2 (therapy): the woundwas washed with 50 μg of ASO 1537S (8 mice in group) (Sequence of theoligonucleotide provided in Example 1) in 200 μl. After suturing, abolus of 250 μl containing 50 μg of Control Oligo 154 (6 control mice)or 250 μl containing 50 μg of ASO Oligo 1537S was applied (8 therapymice). Two days later mice received 6 injections with Control. Oligo 154or ASO Oligo 1537S every other day. The first injection wasintraperitoneal and the second injection was in the tail vein.

On day 30 after surgery, the 6 mice treated with Control Oligo 154 hadtumors on the order of 2000 mm3 and were sacrificed (FIG. 15, circles).On day 57 after surgery, 4 out of the 8 mice receiving ASO Oligo 1537Swere without tumors (FIG. 15, triangles). On day 17 post surgery, 4 outof the 8 mice receiving ASO Oligo 1537S began to show small tumors thatgrew slowly which at day 36 reached a size of about 500 mm3 (FIG. 15,squares). These mice were again subjected to surgery to remove thetumors, and subsequently 2 mice died. The other 2 mice were subjected tothe same therapeutic protocol (alternating 3 injections intraperitonealand 3 injections intravenous) with 50 μg of ASO Oligo 1537S in 250 μl(FIG. 15).

1-64. (canceled)
 65. A method for treating or suppressing metastaticcancer in an individual comprising administering to the individual aneffective amount of one or more oligonucleotide complementary to a humannon-coding chimeric mitochondrial RNA molecule comprising: a. anantisense 16S mitochondrial ribosomal RNA covalently linked at its 5′end to the 3′ end of a polynucleotide with an inverted repeat sequenceor b. a sense 16S mitochondrial ribosomal RNA covalently linked at its5′ end to the 3′ end of a polynucleotide with an inverted repeatsequence, wherein the one or more oligonucleotide is able to hybridizewith the chimeric mitochondrial RNA molecules to form a stable duplex,wherein the metastatic cancer is a non-solid cancer selected from thegroup consisting of multiple myeloma, acute myeloid leukemia, acutelymphoblastic leukemia, chronic myelogenous leukemia, chroniclymphocytic leukemia, acute nonlymphocytic leukemia, acute granulocyticleukemia, chronic granulocytic leukemia, acute promyelocytic leukemia,adult T-cell leukemia, aleukemic leukemia, leukocythemic leukemia,leukemia bovine leukemia, chronic myelocytic leukemia, leukemiacomplexion, embryonal leukemia, eosinophilic leukemia, Gross' leukemia,hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia,histiocytic leukemia, stem cell leukemia, acute monocytic leukemia,leukopenia leukemia, leukopenia lymphocymia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, stem cell leukemiaidiopathic myelofibrosis, Non-Hodgkin's lymphoma, Hodgkin's lymphoma,and myelodysplastic syndrome, and wherein the individual has beenpreviously treated for cancer with a therapy.
 66. The method of claim65, wherein the individual has been previously treated for cancer with atherapy selected from the group consisting of chemotherapy, radiationtherapy, surgery, and combinations thereof
 67. The method of claim 65,wherein the one or more oligonucleotide is complementary to theantisense 16S mitochondrial ribosomal RNA molecule encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:203, SEQ ID NO: 204, SEQ ID NO:
 205. 68. The method of claim 65, whereinthe one or more oligonucleotide comprises a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 7-198.
 69. The method of claim65, wherein the one or more oligonucleotide comprises a nucleotidesequence selected from the group consisting of SEQ ID NOs: 36, 54, 61,65, 70, 74, 76, 79, 81, 88, 197, and
 198. 70. The method of claim 69,wherein the non-solid cancer is selected from the group consisting ofmultiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia,chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy-cellleukemia, hemoblastic leukemia, stem cell leukemia, acute monocyticleukemia, leukopenia leukemia, lymphocytic leukemia, lymphogenousleukemia, lymphoid leukemia, Non-Hodgkin's lymphoma, and Hodgkin'slymphoma.
 71. The method of claim 65, wherein the non-solid cancer isselected from the group consisting of multiple myeloma, leukemia, andlymphoma
 72. The method of claim 71, wherein the one or moreoligonucleotide comprises a nucleotide sequence of SEQ ID NO:36.
 73. Themethod of claim 71, wherein the one or more oligonucleotide comprises anucleotide sequence of SEQ ID NO:
 54. 74. The method of claim 71,wherein the one or more oligonucleotide comprises a nucleotide sequenceof SEQ ID NO:
 61. 75. The method of claim 71, wherein the one or moreoligonucleotide comprises a nucleotide sequence of SEQ ID NO:
 65. 76.The method of claim 71, wherein the one or more oligonucleotidecomprises a nucleotide sequence of SEQ ID NO:
 70. 77. The method ofclaim 71, wherein the one or more oligonucleotide comprises a nucleotidesequence of SEQ ID NO:
 74. 78. The method of claim 71, wherein the oneor more oligonucleotide comprises a nucleotide sequence of SEQ ID NO:76.
 79. The method of claim 71, wherein the one or more oligonucleotidecomprises a nucleotide sequence of SEQ ID NO:
 79. 80. The method ofclaim 71, wherein the one or more oligonucleotide comprises a nucleotidesequence of SEQ ID NO:
 81. 81. The method of claim 71, wherein the oneor more oligonucleotide comprises a nucleotide sequence of SEQ ID NO:88.
 82. The method of claim 71, wherein the one or more oligonucleotidecomprises a nucleotide sequence of SEQ ID NO:
 197. 83. The method ofclaim 71, wherein the one or more oligonucleotide comprises a nucleotidesequence of SEQ ID NO:
 198. 84. A kit comprising one or moreoligonucleotides complementary to an antisense non-coding chimericmitochondrial RNA (ASncmtRNA) molecule or a sense non-coding chimericmitochondrial RNA (SncmtRNA) molecule and instructions for practicingthe methods of claim 65.